3 - azidomethyl - 7-(alpha-aminophenyl(or thienyl)acetamido)ceph - 3 -em-4-oic acids

ABSTRACT

3-AZIDOMETHYL - 7 - (A-AMINOPHENYL(OR THIENYL)ACETAMIDO) CEPH-3-EM-4-OIC ACIDS AND THEIR NONTOXIC, PHARMACEUTICALLY ACCEPTABLE SALTS ARE VALUBALE AS ANTIBACTERIAL AGENTS, AS NUTRITIONAL SUPPLEMENTS IN ANIMAL FEEDS AND AS THERAPEUTIC AGENTS IN POULTRY AND ANIMALS, INCLUDING MAN, AND ARE ESPECIALLY USEFUL IN THE TREATMENT OF INFECTIOUS DISEASES CAUSED BY GRAM-POSITIVE AND GRAM-NEGATIVE BACTERIA. 3-AZIDOMETHYL-7-(A-AMINOPHENYLACETAMIDO) CEPH-3-EM-4-OIC ACID IS PREFERRED EMBODIMENT OF THE INVENTION.

United States Patent 3,634,418 3 AZIDOMETHYL 7-(a-AMINOPHENYL[ORTNYHACETAMIDWCEPH 3 EM-4-0IC ACIDS David Willner, Dewitt, N.Y., assignorto Bristol-Myers Company, New York, N.Y.

No Drawing. Continuation-impart of application Ser. No. 818,065, Apr.21, 1969. This application Aug. 20, 1969, Ser. No. 851,735

Int. Cl. (107d 99/24 US. Cl. 260-243 C 20 Claims 3 azidomethyl 7(a-aminophenyHor thienyljacetamido)ceph-3-em-4-oic acids and theirnontoxic, pharmaceutically acceptable salts are valuable asantibacterial agents, as nutritional supplements in animal feeds and astherapeutic agents in poultry and animals, including man, and areespecially useful in the treatment of infectious diseases caused bygram-positive and gram-negative bacteria. 3 azidomethyl 7(a-aminophenylacetamido)ceph-3-em-4-oic acid is a preferred embodimentof the invention.

CROSS-REFERENCE TO COPENDING APPLICATION This application is acontinuation-in-part of my prior, copending application Ser. No. 818,065filed Apr. 21, 1969 and now abandoned.

BACKGROUND OF THE INVENTION (1) Field of the invention Thecephalosporins of the present invention possess the usual attributes ofsuch antibacterial agents and are particularly useful because they arewell absorbed upon oral administration.

(2) Description of the prior art Cephalothin and cephaloridine arewell-known antibacterial agents. The literature also containsconsiderable data on the activity of cephaloglycin and cephalexin. Theliterature on cephalosporins has been reviewed by E. P. Abraham, Quart.Rev. (London), 21, 231 (1967), E. Van Heyningen, Advan. Drug Res, 4, 170(1967), and briefly by L. C. Cheney, Annual Reports in MedicinalChemistry, 1967, Academic Press Inc., 111 Fifth Avenue, New York, N.Y.,10003, on pages 96 and 97.

The preparation of various 7-[u-amino-arylacetamido]- cephalosporanicacids and the corresponding desacetoxy compounds in which arylrepresents unsubstituted or substituted phenyl or 2- or 3-thienyl isdescribed in British specifications 985,747, 1,017,624, 1,054,806 and1,123,333, in Belgian Patent 696,026 (Farmdoc No. 29,494), in U.S. Pats.3,311,621 and 3,352,858, in Japanese Pat. 16,871/66 (Farmdoc No.23,231), by Spencer et al., J. Med. Chem., 9 746-750 (1 966) and byKurita et al., J. Antibiotics (Tokyo) (A) 19, 243249 (1966).

The preparation of various 3-azidomethyl-7-acylamidoceph-3-em-4-oicacids, but none of the compounds of the present invention, is describedin US. Pats. 3,274,186; 3,278,531 and 3,360,515 and in British patentspecifications 1,012,943; 1,057,883; 1,074,075; 1,104,937 and 1,109,525and in Dutch specifications 66/06820 (Farmdoc No. 23,984); 67/17107(Farmdoc No. 32,566); 67/14888 (Farmdoc No. 31,936); 68/00751 (FarmdocNo. 33,079); and 68/ 04140 (Farmdoc No. 34,027) and in Belgian Pat.714,518 (Farmdoc No. 35,307).

The preparation of 3-aZidomethyl-7-aminoceph-3-em- 4-oic acid isdescribed in British patent specifications 1,101,422 and 1,104,938.

3,634,418 Patented Jan. 11, 1972 ice SUMMARY OF THE INVENTION Thisinvention comprises antibacterial agents of the formula wherein AI isZ-thienyl, 3-thienyl or in which each of R and R is hydrogen, nitro,di(lower) alkylamino, (lower) alkanoylamino, amino, hydroxy,(lower)alkanoyloxy, (lower)alkyl (comprising straight and branched chainsaturated aliphatic groups having from one to six carbon atomsinclusive), (lower) alkoxy, sulfarnyl, chloro, iodo, bromo, fluoro ortrifluoromethyl; and the nontoxic carboxylic acid salts thereof,including nontoxic metallic salts such as sodium, potassium, calcium andaluminum, the ammonium salt and substituted ammonium salts, e.g. saltsof such nontoxic amines as trialkylarnines, including triethylamine,procaine, dibenzylamine, N-benzyl-beta-phenethylamine, 1 ephenamine, N,Ndibenzylethylenediamine, dehydroabietylamine, N,Nbis-dehydroabietylethylenediamine, N (lower) alkylpiperidine, e.g.,N-ethylpiperidine and other amines which have been used to form saltswith benzylpenicillin; and the nontoxic acid addition salts thereof(i.e., the amine salts) including the mineral acid addition salts suchas the hydrochloride, hydrobromide, hydroiodide, sulfate, sulfamate andphosphate and the organic acid addition salts such as the maleate,acetate, citrate, oxalate, succinate, benzoate, tartrate, fumarate,malate, mandelate, ascorbate and the like.

In these compounds the carbon bearing the free amino group is anasymmetric carbon atom and thus the reagents and final products canexist in two optically active, isomeric forms (the D'- andL-diastereoi-somers) as well as in a mixture of the two optically activeforms, all of which are included in the present invention. It should benoted in connection with the foregoing consideration of thediastereoisomers of this invention that isomers other than the twocaused by the asymmetric carbon of the side chain are possible due tothe presence of asymmetric carbon atoms in the 7-aminocephalosporanicacid nucleus. Such additional isomers, however, are not presentlysignificant since 7-aminocephalosporanic acid which is the product offermentation processes is consistently of one configuration; such7-aminocephalosporanic acid is presently used in the production of thecompounds of this invention.

The compounds of the present invention are prepared by either of twomethods. In the equations Ar has the meaning set out above, B representsa standard blocking agent for an amino group, e.g. t-butoxycarbonyl, andthe structure is represented as -Amcephem.

The first synthesis proceeds as follows:

i Ar $11- U-O II i? 1 plus condensing agent Ar H U-Amcephem- C HzNs (I?1 plus agent to remove 13 .ArCH-UAmcephcmOHzNs NHz The second synthesisproceeds as follows:

In the treatment of bacterial infections in man, the compounds of thisinvention are administered topically, orally and parenterally inaccordance with conventional procedures for antibiotic adminstration inan amount of from about to 125 mg./kg./ day and preferably in the rangeof 15 to 50 mg./kg./day in divided dosages, e.g., three or four times aday. They are administered in dosage units containing, for example 125,250, 500, 1000 and 2000 mg. of active ingredient with suitablephysiologically acceptable carriers or excipients. The dosage units canbe in the form of liquid preparations such as solutions, dispersions,emulsions or in solid form such as tablets, capsules, etc.

The following examples are given in illustration of, but not inlimitation of, the present invention. All temperatures are in degreescentigrade. Skellysolve B is a petroleum ether fraction of B.P. 60-68 C.consisting essentially of n-hexane.

DESCRIPTION OF THE PREFERRED EMBODIMENTS EXAMPIJE 1 1 COOH magnesiumoxide powder was intimately mixed by grinding in a mortar. The mixturewas suspended in 500 ml. of 50% aqueous dioxane and 58.0 g. (0.4 mole)of t-butoxycarbonylazide (commercial sample from Aldrich,t-butoxycarbonyl chloride, t-butylchloroformate, is reported to beunstable above 10 C.) was added dropwise with vigorous stirring. Thereaction mixture was heated with stirring for 20 hours at 45-50. Thecoled mixture was then diluted with 2 l. of ice-water and 500 ml. ofethyl acetate with stirring. A small amount of solid was removed byfiltration and the filtrate was separated into its component layers. Theorganic phase was extracted with 150 ml. portions of 3% sodiumbicarbonate solution and water. These extracts were combined with theaqueous phase above and the solution was cooled and acidified to pH 5.(It is presumed that lowering the pH further would result in theliberation of hydrazoic acid.) The product was extracted into ethylacetate (3X 250 ml.) and the combined extracts were washed with waterand dried over magnesium sulfate. Evaporation of the solvent gave an oilwhich was solidified by trituration with Skellysolve B and ether. Thisprocedure afforded 29.5 g. D()-a-t-butoxycarboxamidophenylacetic acid asWhite crystals of M.P. 88-90" dec.; [a] 135 (0:1.0, OHgOH). The motherliquors afforded a second crop of 10.5 g., M.P. 8688. Total yield 40.0g.,

3 azidomethyl 7 (D 0c arninophenylacetamido)- ceph-3-em-4-oic acid-5 .02grams of t-butoxycarbonyl-D- phenylglycine was dissolved in 80 ml. ofdry tetrahydrofuran. 2.8 ml. of triethylamine were added and the mixturestirred and cooled to 10. At that temperature 2.6 ml. of isobutylchloroformate was added dropwise and the mixture was stirred for anadditional 10 minutes at 10. A solution of 5.1 grams of7-amino-3-azidomethylceph-3-em-4-oic acid in 72 ml. of 50% aqueoustetrahydrofuran containing 2.8 ml. of triethylamine was added to themixed anhydride solution. The mixture was stirred for one hour whilecooling it with an ice bath and an additional hour without externalcooling. The tetrahydrofuran was evaporated at reduced pressure and theresidue was treated with a mixture of 120 ml. water and 40 ml. ethylacetate. The ethyl acetate was discarded. The aqueous layer was coveredwith 120 ml. of ethyl acetate, chilled and acidified to pH 2.5 with 10%HCl. The emulsion obtained was filtered through filter aid and the ethylacetate layer separated. The extraction was repeated and the combinedorganic solutions were washed with water, dried over Na SO and thesolvent removed under reduced pressure. There remained a foam weighing 5grams. Its IR spectrum showed strong absorption at 2112 cm? (azide) and1785 cm.- (fi-lactam). The foam was dissolved in 50 ml. oftrifluoroacetic acid at 4. After 3 minutes the solution was poured into1000 ml. of dry ether to precipitate the trifluoroacetic acid salt of3-azidomethyl-7-(D 0c aminophenylacetamido)ceph- 3-ern-4-oic acid whichwas collected by filtration and then was stirred for one hour in amixture of 20 ml. of water and 10 ml. of 25% Amberlite LA-l resin(acetate form) in isobutyl methyl ketone. [The preparation of the resinis described in J. Med. Chem., 9, 746 (1966).] The clear aqueous layerwas separated and extracted 3 times with isobutyl methyl ketone and oncewith ether. The aqueous solution was concentrated at reduced pressureand at a temperature not exceeding 40 to a small volume (whenprecipitation occurred) and was kept overnight in the refrigerator. mg.3-aZidomethyl-7-(D-aaminophenylacetamido)ceph 3 em 4 oic acid werecollected by filtration the next day. Its IR was consistent with theabove structure having major peaks at 2110 (azide); 1780 (fi-lactam);1700, 1530 (amide); 1615 (carboxylate ion).

This compound, after conversion to its sodium salt by solution in 5%NaHCO was found in duplicate experiments to exhibit the followingMinimum Inhibitory Concentrations (MIC) in meg/ml. versus the indicatedmicroorganisms as determined by overnight incubation at 37 C. inNutrient Broth by serial tube dilution:

Organism M.I.C. meg/ml. D. pneumoniae plus serum .080.6 S. aureus Smith.l60.6 S. aureus Smith plus 50% serum 2.5-5 S. aureus 1633-2 .6 Pr.morganii 63 E. coli A9675 4 E. coli Juhl A15119 2-4 Sal. enteritidis 1.3K. pneumoniae A9977 2.5-1.3 K. pneumoniae Al5130 4 Ps. aeruginosa500-500 Pr. mirabilis A9900 1.3 S. marcescens A20019 125-250 EXAMPLE 2D()) a t butoxycarboxamidophenylacetic acid. A mixture of 30.0 g. (0.2mole) D()-2-phenylglycine (also called D-a-aminophenylacetic acid) and 16.0 g. (0.4 mole) of magnesium oxide powder was intimately mixed bygrinding in a motar. The mixture was suspended in 500 ml. of 50% aqueousdioxane and 58.0 g. (0.4 mole) of t-butoxycarbonyl azide (commercialsample from Aldrich, t-butoxycarbonyl chloride, t-butylchloroformate, isreported to be unstable above was added dropwise with vigorous stirring.The reaction mixture was heated with stirring for hours at 45-50. Thecooled mixture was then diluted with '2 l. of ice-water and 500 ml. ofethyl acetate with stirring. A small amount of solid was removed byfiltration and the filtrate was separated into its component layers. Theorganic phase was extracted with 150 ml. portions of 3% sodiumbicarbonate solution and Water. These extracts were combined with theaqueous phase above and the solution was cooled and acidified to pH 5(it is presumed that lowering the pH further would result in theliberation of hydrazoic acid.) The product was extracted into ethylacetate (3X 250 ml.) and the combined extracts were washed with waterand dried over magnesium sulfate. Evaporation of the solvent gave an oilwhich was solidified by trituration with Skellysolve B and ether. Thisprocedure afforded 29.5 g. D()-ot-t-butoxycarboxamidophenylacetic acidas white crystals of M.P. 88-90 dec.; [at] 135 (c. =l.0, CH OH). Themother liquors afforded a second crop of 10.5 g., M.P. 86-88. Totalyield 40.0 g., 80%.

7 [D a t butoxycarboxamidophenylacetamido] cephalosporanic acid.-(J. L.Spencer et al., J. Med. Chem., 9, 746 (1966).) A solution of 42.7 g.(0.17 mole) of D()-t-butoxycarboxamidophenylacetic acid and 17.2 g.(0.17 mole) of triethylamine in 600 ml. of tetrahydrofuran (TI-IF) wascooled to 10. 18.5 g. (0.17 mole) of ethyl chloroformate was addeddropwise with stirring, and the mixture was stirred for 10 min. at 10.To the mixed anhydride solution thus formed was added in one portion acold (0) solution of 46.3 g. (0.17 mole) of 7 aminocephalosporanic acidand 17 .2 g. (0.17 mole) of triethylamine in 600 ml. of 50% aqueous THF.The mixture was stirred for 1 hour at 0-5, and the THF was then removedunder reduced pressure. (Any solid material at this stage should beremoved by filtration.) The residue was dissolved in a mixture of 1 l.of cold water and 400 ml. of ethyl acetate, and after a brief shakingthe layers were separated. The aqueous phase was cooled, layered withethyl acetate and 42% phosphoric acid was added to pH 3 with stirring.The phases were separated and an extraction was made with fresh ethylacetate. The combined extracts were washed with cold water, dried overmagnesium sulfate and the solvent was removed under reduced pressure.After trituration with ether and Skellysove B the residual oilsolidified to give 62.5 g. (72.5%) of7-[D-ot-t-butoxycarboxamidophenylacetamido]cephalosporanic acid.

3 azidomethyl-7-(D-ot-aminophenylacetamido)ceph-3- em-4-oicacid.7-(D-ot-t-butoxycarboxamidophenylacetamido)cephalosporanic acid (25grams) was dissolved in ml. of water and 50 ml. of 1 N NaHCO The pH ofthe solution was adjusted to 8 with a 2 N Na CO solution. Ten grams ofsodium azide were added and the solution kept at 50 C. for 17 hours. Thesolution was chilled, layered with ethyl acetate and acidified withcone. HCl to pH 2. The organic layer was separated and the extractionrepeated. The combined organic solutions were washed with water, driedover Na SO and the solvent evaporated. The foam that was left had thecorrect IR with absorption at 2100 (azide) and 1785 (ti-lactam). Fivegrams of the foam were dissolved in ml. of 50% aqueous formic acid. Thesolution was heated at 40 for 3 hours. The solvent was evaporated andthe residue treated with toluene and evaporated again. The oily residuewas triturated with a mixture of 56 ml. ethyl acetate and 3.8 ml. water.The crystalline solid,3-azidomethyl-7-(D-uaminophenylacetamido)ceph-3-em-4-oic acid, obtainedwas collected by filtration and weighed 2.89 grams. Its IR spectrum wasthe same as that described in Example 1 and consistent with structure.

EXAMPLE 3 Substitution in each of the procedures of Examples 1 and 2forthe D-a-aminophenylacetic acid used therein of an equimolar weight ofD-a-amino-2-thienylacetic acid, D-u-amino-3-thienylacetic acid,D-tx-amnio-m-nitrophenylacetic acid, D-ot-amino-M-aminophenylaceticacid, DL-ot-amino-p-methylphenylacetic acid,DL-a-amino-m-methylphenylacetic acid, DL-a-amino-p-chlorophenylaceticacid, DL-a-amino-m-chlorophenylacetic aicd,DL-ot-amino-p-methoxyphenylacetic acid, DL-u-amino-m-methoxyphenylaceticacid, DL-a-amino-p-fiuorophenylacetic acid,DL-a-amino-m-fluorophenylacetic acid, DL-a-amino-p-hydroxyphenylaceticacid, DL-tx-amino-m-hydroxyphenylacetic acid,DL-a-amino-p-aminophenylacetic acid, DL-a-amino-m-aminophenylaceticacid, DL-a-amino-m-nitrophenylacetic acid,DL-ot-amino-p-dimethylaminophenylacetic acid,DL-a-amino-m,p-dimethoxyphenylacetic acid, D-a-amino-m-iodophenylaceticacid, D-rx-amino-m-chloro-p-hydroxyphenylacetic acid,D-ot-amino-p-methoxyphenylacetic acid, D-vt-amino-p-hydroxyphenylaceticacid, D-a-amino-m-methoxyphenylacetic acid,D-u-amino-m-hydroxyphenylacetic acid, D-ot-amino-p-acetamidophenylacticacid, D-a-amino-m-aminophenylacetic acid, and

D-wamino-m-acetamidophenylacetic acid, respectively, produces3-azidomethyl-7- (D-ot-2-thienylacetamido) ceph-3-em-4- oic acid,3-azidomethyl-7-(D-ot-3-thienylacetamido)ceph-3-em-4- oic acid,

3-azidomethyl-7- (D-ot-m-nitrophenylacetamido ceph-3- em-4-oic acid,

3-azidomethyl-7-(D-a-m-aminophenylacetamido)ceph-3- em-4-oic acid,

3-azidomethyl-7-(DL-u-p-methylphenylacetamido)ceph- 3-em-4-oic acid,

3 -azidomethyl-7-( DL-a-m-methylphenylacetamido ceph 3-em-4-oic acid,

3-azidomethyl-7-(DL-u-p-chlorophenylacetamido)ceph-3- em-4-oic acid,

3-azidomethyl-7- (DL-ot-m-chlorophenylacetamido) ceph- 3-em-4-oic acid,

3-azidomethyl-7- (DL-ot-p-methoxyphenylacetamid'o) ceph- 3-em-4-oicacid,

3-azidomethyl-7- (DL-u-m-methoxyphenylacetamido) ceph-3-em-4-oic acid,

3-azidomethyl-7- (DL-u-p-fiuorophenylacetamido ceph- 3-em-4-oic acid,

3-azidomethyl-7- (DL-ot-m-fluorophenylacetamido) ceph- 3-em-4-oic acid,

3-aZidomethyl-7- (DL-ot-p-hydroxyphenylacetamido ceph- 3-em-4-oic acid,

3 -azidomethyl-7- (DL-a-m-hydroxyphenylactamido) ceph- 3-em-4-oio acid,

3-azidomethyl-7- (DL-a-p-aminophenylacetamido ceph-3- em-4-oic acid,

3-azidomethyl-7 (DL-a-p-arninophenylacetamido) ceph-3 em-4-oic acid,

3-azidomethy1-7- DL-u-m-nitrophenylacetamido ceph-3- em-4-oic acid,

3 -azidomethyl-7- DL-a-p-dirnethylaminophenylacetamido ceph-3-em-4-oicacid,

3-azidomethyl-7- (DL-ot-m,p-dimethoxyphenylacetamido) ceph-3-em-4-oicacid,

3-azidomethyl-7- (D'a-m-iodophenylacetamido ceph-3- em-4-oic acid,

3-azidomethy1-7- (D-m m-chloro-p-hydroxyphenylacetamido) ceph-3-em-4-oicacid,

3-azidomethyl-7- (D-a-p-methoxyphenylacetamido ceph- 3-em-4-oic acid,

3-azidomethyl-7- (D-ot-p-hydroxyphenylacetamido ceph- 3-em-4-oic acid,

3-azidomethyl-7- (D-u-m-methoxyphenylacetamido) ceph- 3-em-4-oic acid,

3-azidomethyl-7- (D-a-m-hydroxyphenylacetamido ceph- 3-em-4-oic acid,

3 -azidomethyl-7- (D-a-p-acetamidophenylacetamido) ceph-3-em-4-oic acid,

3-azidomethyl-7- (D-a-m-aminophenylacetamido) ceph- 3-em-4-oic acid, and

3-azidomethyl-7- (D-a-m-acetamidophenylacetamido) cepl1-3-em-4-oic acid,respectively.

The preparation of each of these a-amino acids is described in eitherUS. Patents 3,198,803 and 3,342,677 or by Friis et al., Acta Chem.Scand. 17, 2391-2396 (1966) or by Neims et al., Biochemistry (Wash) 5,203- 213 (1966) or in the section below entitled IllustrativePreparations of Starting Reagents.

EXAMPLE 4 3-azidomethyl 7 (2,2-dimethyl 4phenyl-S-oxo-limidazolidinyl)ceph-3-em-4-oic acid-1.09 grams of 3-aZidomethyl-7- D-a aminophenylacetamido ceph-3 -em- 4-oic acid aresuspended in a mixture of 60 ml. dry methanol and 100 ml. dry acetone.The mixture is stirred and 280 mg. of triethylamine in 20 ml. of drymethanol are added. A clear solution is obtained and it is stirred for19 hours. The solvent is evaporated under reduced pressure and theresidue is treated with 20 ml. of cold water and 20 ml. of ethylacetate. The mixture is acidified to pH 3 and shaken well. The emulsionthus obtained is filtered through Celite and the organic layerseparated. The aqueous phase is saturated with NaCl and extracted twicemore with ethyl acetate. The combined ethyl acetate fractions are washedwith water and, without drying, the solvent is evaporated. The residueis triturated with dry ether and filtered. The dry solid weighs 240 mg.Its IR and NMR are consistent with structure.

EXAMPLE 3-azidomethyl-7-(D-u-amino 3 thienylacetamido) ceph-3-em-4-oicacid.-Five grams (.02 mole) D-t-butoxycarbonyl-3-thienylglycine (alsocalled D-()-a-t-butoxycarboxamido-3-thienylacetic acid) are dissolved in100 ml. methylene chloride containing 2.02 g. (.02 mole) triethylamineat -5 to C. With vigorous stirring and cooling to 5 to -10 C., 2.4 g.pivalyl chloride in 15 ml. methylene chloride are added dropwise. Themixture is stirred for 2 hours at -5 to 10 C. A par tial suspension of5.1 g. (.02 mole) 7-amino-3-azidomethylceph-3-em-4-oic acid in ml.methylene chloride containing 6.06 g. (.06 mole) triethylamine is addedportionwise keeping the temperature below 5 C. The icesalt bath isimmediately replaced with a regular ice-bath and the mixture stirredvigorously at 3 C. for 3 hours. The methylene chloride is thenflash-evaporated and the residue taken up in 100 ml. water and 100 ml.ethyl acetate. The mixture is stirred while being acidified to pH 2.5with 10% HCl. The emulsion is filtered through Celite and the phases areseparated. The aqueous phase is extracted with 100 ml. ethyl acetate andthe combined organic phases washed with water, dried over Na SO andflash-evaporated. The residue is triturated with ether and 'cyclohexaneto yield 5.5 g. solid after drying. This is taken up in 40 ml. 3:1 ethylacetate-ether and filtered in 500 ml. cyclohexane with stirring forreprecipitation. The solid is collected, washed with cyclohexane anddried under high vacuum. Yield 4.1 g. TLC shows little or notboc-3-thienylglycine. IR consistent with structure showing N ,B-lactam,amide and carboxylic acid. The material is added to a stirred, cooled 35ml. volume of trifluoroacetic acid. After solution occurs, the mixtureis stirred for 4 minutes. The mixture is then poured into 750' ml. 2:1Skellysolve B-ether for precipitation. The solid is collected and washed3 times with Skellysolve B. The material is then added to a mixture of50 ml. Amberlite LA-l resin (acetate form) in MIBK and 100* ml. water.This mixture is stirred for 1 hour. The layers are separated, theaqueous layer washed twice with ether and flash-evaporated. The residueof 3-azidomethyl-7-(D-w amino-3-thienylacetamido)ceph-3-em-4-oic acid istriturated with isopropanol, filtered, washed with dry ether and dried.It weighs 1.4 g. Its IR and NMR are consistent with structure.

EXAMPLE 6 3 azidomethyl-7-(D-a-arnino 2 thienylacetamido)ceph-3-em-4-oic acid.-2.8 ml. (.02 mole) triethylamine are added to asolution of 5.2 g. (.02 mole) D-tbutoxycarbonyl-2-thienylglycine in 80'ml. dry tetrahydrofuran with vigorous stirring and cooling below 10 C.2.6 mls. (.02 mole) isobutyl chloroformate are added dropwise withcontinued stirring and cooling below 1() C. The mixture is stirred atthis temperature for 12 minutes at the end of which time 5.1 g. (.02mole) 7-amino-3- azidomethylceph-3em-4-oic acid in a solution of 72 ml.50% aqueous tetrahydrofuran containing 5.6 ml. (.04 mole) triethylamineare added. The ice-salt bath is immediately replaced with a regular icebath and the mixture is stirred at 0 C. for 1 hour. The mixture is thenstirred for an additional hour at room temperature. The tetrahydrofuranis flash-evaporated and the residue treated with 15 0 ml. water and ml.ethyl acetate. The phases are separated and the aqueous portionextracted 3 more times with ethyl acetate. The combined organic extractsare washed with 5% H01 solution. The organic solution is then washedwith water, dried over Na SO filtered and flash-evaporated. The residueis triturated with ether and cyclohexane to give a solid which iscollected by filtration, redissolved in a small volume of ether andreprecipitated by adding cyclohexane. The material is collected, washedwith cyclohexane and dried. The material is then dissolved in 30 ml. 45%formic acid and stirred at 40 C. for 3 hours. The mixture is thenflash-evaporated, azeotroped 3 times with toluene and triturated withwet ethyl acetate to yield 500- mg. of 3-azidomethyl-7-(D-u-amino-2-thienylacetamido)ceph-3-em-4-0ic acid. M.P. decomp, at 230 C. Infraredabsorption spectrum consistent for structure, shows bands for NB-lactam, amide and carboxylate at 2100, 1760, 1680, 1595 and 1403 cm.-

9 EXAMPLE 7 3 azidon1ethyl-7-(D-ot-aminophenylacetamido)ceph-3- em-4-oicacid-Under anhydrous conditions, 12.55 g. (.05 mole)D-t-butoxycarbonyl-2-phenylglycine are dissolved and cooled to 10 C. in400 m1. methylene chloride (MeCl containing 5.05 g. (.05 mole)triethylamine. Vigorous stirring and cooling between 5 and 10 C. aremaintained as 6.05 g. (.05 mole) pivalyl chloride in 100 ml. MeCl areadded dropWise. The mixture is then stirred at -5 to 10 C. for 2 hours.Meanwhile, a chilled suspension of 12.8 g.7-amino-3-azidomethyl-ceph-3-em-4oic acid in 500 ml. drydimethylformamide (D MF) is treated portionwise with 27.8 g. (.15 mole)tributylamine and stirred until most of the solid is in solution. Thismixture is then added all at once to the mixed anhydride reactionmixture. The icesalt bath is immediately replaced with a regular icebath and the mixture is stirred vigorously at to 3 C. for 3 hours. Thesolvents are removed at reduced pressure (aspirator and high vacuum) ata temperature not higher than 35 To the remaining residue 500 ml. waterand 500 ml. ethyl acetate are added and the mixture is shaken vigorouslyuntil the residue has dissolved. The phases are separated (by filtrationthrough Celite if necessary) and the aqueous phases extracted 4 timeswith 250 ml. ethyl acetate. The combined organic phases are washed with5% HCl until the wash solution remains acidic. The ethyl acetatesolution is then washed twice with water and dried over Na S'O Thedrying agent is filtered off and the solution flash-evaporated. Theresidue is triturated with ether and cyclohexane to give a yellow ishsolid which is collected, washed with cyclohexane and dried in open air.The material is dissolved in 100 ml. 3:1 ethyl acetate-ether solution,filtered and reprecipitated by adding 2 liters of cyclohexane. The solidis collected, washed with cyclohexane and dried in open air to yield12.8 g. of 3-aZidomethyl-7-[Da-(t-butoxycarbonylamido)phenylacetamido]ceph-3-em-4-oic acid. 23.6grams of this compound are suspended in 680 ml. of 45% formic acid. Themixture is stirred and heated for 3 hours at 40. The solvent isflash-evaporated and the residue is azeotroped 3 times with 300 ml. oftoluene. The residue is triturated with wet ethyl acetate to give asolid which is collected, washed with ethyl acetate and dry ether andthen dried at high vacuum to yield 16.1 grams of3-aZidomethyl-7-(D-a-aminophenylacetamido)- ceph-3-em-4-oic acid. [a+82.4 (C, 0.5 in 50% aqueous acetonitrile).

EXAMPLE 8 3-azidomethyl 7 (D-a-aminophenylacetamido)ceph- 3-em-4-oicacid.5 g. (.02 mole) D-t-butoxycarbonyl- 2-phenylglycine are dissolvedin 100 ml. methylene chloride containing 2.02 g. (.02 mole)triethylamine at -5 to -10 C. With vigorous stirring and cooling to -5C. to 10C., 2.4 g. (.02 mole) pivaloyl chloride in ml. MeCl are addeddropwise. The mixture is stirred at -5 to -10 C. for 2 hours. A partialsuspension of 5.1 g. (0.02 mole) 7-amino-3azidomethyl-ceph-3em-4- oicacid in 100 ml. chloroform is treated with 6.0 6 g. (.06 mole)triethylamine With cooling to 5 C. This mixture is added portionwise tothe reaction mixture above, keeping the temperature below 5 C. Theice-salt bath is immediately replaced with a regular ice-bath and themixture is stirred for 3 hours at 3 C. The solvents are thenflash-evaporated and the residue taken up in 100 ml. water and 100 ml.ethyl acetate. This mixture is stirred and chilled while being acidifiedto pH 2.5 With 10% HCl. The phases are separated, the aqueous phaseextracted with 100 ml. ethyl acetate and the combined organic phaseswashed with water, dried over Na SO and flash-evaporated. The residue istriturated with ether and cyclohexane to yield 4 g. of3-aZidomethyl-7-[- or- (t-butoxycarboxamido)phenylacetamido]ceph 3 em-4-oic acid. Its infrared spectrum is consistent with structure. Thet-boc-protective group is removed as in the proceeding example to yield3-aZidomethyl-7-(D-otaminophenylacetamido)ceph-3-em-4-oic acid.

EXAMPLE 9 3-azidomethyl 7 (D-ot-aminophenylacetamido)ceph- 3-em-4-oicacid-5 g. (.02 mole) D-t-butoxycarbonyl-2- phenylglycine are dissolvedin ml. methylene chloride containing 2.02 g. (.02 mole) triethylamine at-5 to l0 C. With vigorous stirring and cooling to 5 C. to l0 C., 2.4 g.pivalyl chloride in 15 ml. methylene chloride are added dropwise. Themixture is stirred for 2 hours at 5 to 10 C. A partial suspension of 5.1g. (.02 mole) 7-amino3-azidomethylceph-3em-4-oic acid in 100 ml.methylene chloride containing 6.06 g. (.06 mole) triethylamine is addedportionwise keeping the temperature below 5C. The ice-salt bath isimmediately replaced with a regular ice-bath and the mixture stirredvigorously at 3 C. for 3 hours. The methylene chloride is thenflash-evaporated and the residue taken up in 100 ml. water and 100 ml.ethyl acetate. The mixture is stirred in ice while being acidified to pH2.5 with 10% HCl. The emulsion is filtered through Celite and the phasesseparated. The aqueous phase is extracted with 100 ml. ethyl acetate andthe combined organic phases washed with Water, dried over -Na SO andflash-evaporated. The residue is triturated with ether and cyclohexaneto yield 5.5 g. solid after drying. This is taken up in 40 ml. 3:1 ethylacetate-ether, and filtered into 500 m1. cyclohexane with stirring forreprecipitation. The solid is collected, washed with cyclohexane anddried under high vacuum. Yield 4.1 g. TLC shows little or noZ-phenylglycine. Infrared absorption spectrum consistent with structureshowing N fi-lactam, amide and carboxylic acid.

The protective group is removed as in Example 7 to yield the titlecompound.

EXAMPLE 10 11 1'1 EOH I'm CH3 (1:0 CH. ('J

VII

IV VII H s 45% HCOOH la ll (13:0 0 N\/CIIzNa 6 600K CH s GHQ VIII IL'HzIii: l

O= N\/ -oH2N IX )0011 Procedure (1) 7-ACA-ptoluenesulphonate dihydrate(1 11). Three hundred grams of crude 7-ACA (7-aminocephalosporanic acid)(I) are suspended and stirred in 750 ml. of acetone (reagent grade). Tothis is added a solution of 360 g. of p-toluenesulphonic acidmonohydrate (II) in 750 ml. of acetone. The mixture is stirred for aboutminutes, during which time most of the material goes into solution. Thesolution is filtered through a sintered glass funnel (grade C) layeredwith diatomaceous earth (Celite) as filter aid. The Celite layer is thenwashed with 225 ml. of acetone (note 1) and this washing is added to thefiltrate. The clear solution is stirred and 120 ml. of distilled waterare added. The solution is now cooled in an ice-salt bath (Note 2).After a short white crystallization starts (Notes 3, 4). The mixture isstirred in the ice-salt bath for 2-4 hours and then filtered. The solidis sucked as dry as possible and then slurried well in 750 ml. of cold(0) acetone. The mixture is filtered and the solid sucked as dry aspossible. The solid is then dried at high vacuum over P 0 The solid,7-ACA-ptoluenesulphonate dihydrate (III), weighs 439 g. (83% yield).

(2) 7-amino-3azidomethylceph-3em-4-oic acid (IV) (Note 5).In a 3000 ml.glass beaker, 53 g. (0.11 moles) of 7-ACA-ptoluenesulphonate dihydrateare suspended in 530 ml. of distilled water. The suspension is stirredefiiciently and a solution of 2 N Na CO is added in small portions untilthe pH is at 6.5-7 (Notes 5, 6). Fifteen grams (0.25 moles) of NaN (Note7) are added to the stirred solution. More carbonate solution is nowadded carefully in small amounts so that all the solid is dissolved andthe pH remains, without drifting, at a pH range of 8-8.2. The solutionis now transferred to a round bottom flask equipped with a stirrer and areflux condenser. The flask is immersed in a water bath heated to 50(Note 8), and the solution is kept at this temperature with stirring for1920 hours. The solution is chilled to 5 and stirred and acidified verycarefully with cold HCl to pH 2.5 (Note 5). The mixture is now stirredin the cold for an additional 10 minutes and is filtered over a sinteredglass funnel, grade C, (Notes 9, 10 as dry as possible. The solid is nowtriturated well with a small amount of ice cold water and sucked dry.This procedure is repeated twice. The solid is now transferred to adesiccator and dried over R 0 at high vacuum (Note 12 11). The drysolid, 7-amino-3-azidomethylceph-3em-4- oic acid, weighs 12.3 g. (46%yield).

(3) D() u t-butoxycarboxamidophenylacetic acid (VII).A mixture of 30.0g. (0.2 mole) of D(-)-2- phenylglycine and 16.0 g. (0.4 mole) ofmagnesium oxide powder was intimately mixed by grinding in a motar. Themixture was suspended in 500 ml. of 50% aqueous dioxane, and 58.0 g.(0.4 mole) of t-butoxycarbonyl azide was added dropwise with vigorousstirring. The reaction mixture was heated with stirring for 20 hours at4550. The cooled mixture was then diluted with 2 l. of ice-water and 500ml. of ethyl acetate with stirring. A small amount of solid was removedby filtration, and the filtrate was separated into its component layers.The organic phase was extracted with 150 ml. portions of 3% sodiumbicarbonate solution and water. These extracts were combined with theaqueous phase above and the solution was cooled and acidified to pH4.5-5. The product was extracted into ethyl acetate (3x250 ml.) and thecombined extracts were washed with water and dried over magnesiumsulfate. Evaporation of the solvent gave an oil which was solidified bytrituration with Skellysolve B and ether. This procedure afforded 29.5g. of D(')at-butoxycarboxamidophenylacetic acid (VII) as white crystalsof MP. 88-90 dec. (reported M.P. 111- 112"); [0d -135 (C, 1.0, CH OH).

The mother liquors afforded a second crop of 10.5 g., M.P. 86-88". Totalyield 40.0 g.,

(4) 3 azidomethyl 7 (D-a-aminophenylacetamido) ceph-3-em-4-oic Acid(IX).Five grams (.02 mole) of VII are dissolved in ml. methylenechloride containing 2.02 g. (.02 mole) triethylamine at 5 to 10- C. Withvigorous stirring and cooling to 5 to 10" C., 2.4 g. pivalyl chloride in15 ml. methylene chloride are added dropwise. The mixture is stirred for2 hours at 5 to 10" C. A partial suspension of 5.1 g. (.02 mole) 7-amino-3azidoceph-3-em-4-oic acid in 100 ml. methylene chloridecontaining 6.06 g. (.06 mole) triethylamine is added portionwise keepingthe temperature below 5 C. The ice-salt bath is immediately replacedwith a regular ice-bath and the mixture stirred vigorously at 3 C. for 3hours. The methylene chloride is then flash-evaporated and the residuetaken up in 100 ml. water and 100* ml. ethyl acetate. The mixture isstirred in ice while being acidified to pH 2.5 With 10% HCl. Theemulsion is fil tered through Celite and the phases separated. Theaqueous phase is extracted with 100 ml. ethyl acetate and the combinedorganic phases are Washed with water, dried over Na SO andflash-evaporated. The oily residue is triturated with ether andcyclohexane to yield 5.5 g. solid after drying (Notes 12, 13). 23.6grams of this compound are suspended in 680' ml. of 45 %formic acid. Themixture is stirred and heated for 3 hours at 40. The solvent isflash-evaporated and the residue is azeotroped 3 times with 300 ml. oftoluene. The residue is triturated with wet ethyl acetate to give asolid which is collected and washed with ethyl acetate and dry ether. Itis then dried at high vacuum to yield 16.1 g. of3-azidomethyl-7-(D-a-aminophenylacetamido)ceph-3-em-4-oic acid. [OLt+82.4- (C, 0.5 in 50% aqueous acetonitrile) (Note 14).

NOTES (1) The amounts of acetone and Water used in this procedure arecritical. If it is found necessary to use more acetone, this should beheld to a minimum.

(2) Do not interrupt stirring.

(3) The start of the crystallization may be delayed sometimes. In such acase, a few cc. of the solution are scratched with a glass rod in a testtube while cooling. The crystals obtained are used as seeds.

(4) As soon as crystallization occurs the mixture thickens and becomescakey. It is advisable to use an efiicient stirrer having a large radiusof stirring.

(5) The successful preparation of this material depends on carefullyfollowing the procedure given. A very accu- 13 rate pH meter should beused, and care should be taken for accurate pH determination,particularly in the 6-8.2 and 2-4 pH regions. Do not overshoot therecommended pH. Extended range pH paper can be used.

(6) The pH tends to drift since not all the 7-ACA salt is in solution.This drift should be kept within the mentioned region by adding morecarbonate solution.

(7) Poison (8) The use of a constant temperature bath heater isrecommended.

(9) It is advised to use a funnel of large capacity so that the solidfiltered will form a thinner layer. This will speed filtration and allowthe solid to be sucked dry easier.

(10) The filtrate contains hydrazoic acid, highly poisonous.

(11) The drying time should be as short as possible.

This can be facilitated by spreading the solid as a thin layer on alarge surface and drying it in this form.

(12) It is advised at this point to check by TLC (thin layerchromatography) to see whether any N-t-butoxycarbonyl-2-phenylglycine ispresent. If it is, the material is taken up in 40 ml. of 3:1 ethylacetate-ether mixture and filtered into 500 ml. of cyclohexane withstirring. The precipitated solid is filtered and dried. The loss isusually 10%.

(13) The reaction described can be scaled up easily, e.g. 4- or 5-fold.

(14) The protective group can also be removed with trifluoroacetic acid.A 20% yield is obtained.

EXAMPLE 1 1 D a t butoxycarbonylamino m acetamidophenylacetic acid.Amixture of 40.3 g. (0.194 mole) of D-aamino-m-acetamidophenylaceticacid, 56.9 g. (0.388 mole) of t-butoxycarbonyl azide, 16.1 g. ofmagnesium oxide, 500 ml. of water and 500 ml. of dioxane was stirredovernight at 45-50". The cooled reaction mixture was poured into 1700ml. of crushed ice-Water that was layered with ethyl acetate and thewhole filtered. The ethyl acetate phase was separated and washed oncewith dilute sodium bicarbonate and this combined with the aqueous phase.The aqueous phase was adjusted to pH 4 with 42% phosphoric acid andextracted 3 times with ethyl acetate. The combined organic extracts werewashed 3 times with water, dried over sodium sulfate, filtered, andstripped of solvent at reduced pressure. The residue was dissolved inanhydrous ether, the solution diluted with Skellysolve B and theprecipitate triturated with Skellysolve B giving a filterable solid;yield 31.6 g. after drying over phosphorus pentoxide.

3 azidomethyl 7 (D onbutoxycarbonylamino-macetamido-phenylacetamido)ceph-3-em-4-oicacid.Ethyl chloroformate (1.54 ml., 0.0162 mole) was added with goodstirring to a solution of 5.0 g. (0.0162 mole) of D-OC-t-butoxycarbonylamino In acetamidophenylacetic acid, 2.38 ml. (0.017mole) of triethylamine and 150 ml. of tetrahydrofuran at 10. The mixturewas stirred at 10 for 25 minutes to form the mixed anhydride. To themixed anhydride thus obtained was added a cold (3) solution of 4.13 g.(0.0162 mole) of 3-azidomethyl-7-arnino-ceph- 3-em-4-oic acid, 4.5 ml.(0.0324 mole) of triethylamine, 75 ml. of tetrahydrofuran and 75 m1. ofwater. The reaction mixture was stirred for 1.5 hours at to About 75 ml.of water was added to the reaction mixture and the tetrahydrofuranstripped off at reduced pressure. The aqueous concentrate was layeredwith ethyl acetate, acidified with 42% phosphorus acid, filtered, thephases separated and the aqueous phase extracted twice more with ethylacetate. The combined organic extracts were washed 3 times with water,dried with sodium sulfate, filtered and the solvent removed at reducedpressure. The residue was triturated with Skellysolve B giving afilterable solid; yield 6.0 g. The solid was dissolved in a combinationof 50 ml. ether plus 175 ml. ethyl acetate and reprecipitated by theaddition of cyclohexane; yield 4.0 g. The solid was then dissolved inabout 40 ml. of chloroform, the solution filtered, and reprecipitatedwith anhydrous ether; yield 2.0 g. The infrared and nuclear magneticresonance spectra Were consistent for 3-azidomethyl-7-(D-a-t-butoxycarbonylamino m acetamidophenylacetamido)ceph 3-em- 4-oic acid.

3 azidomethyl 7 (D-tx-amino-m-acetamidophenylacetamido)-ceph-3-em-4-oicacid.-3 azidomethyl-7-(D- ot-t-butoxycarbonylamino macetarnidophenylacetamido)ceph-3-em-4-oic acid 1.1 g.) was added to 15ml. of trifiuoroacetic acid at 10-15". After addition of an additional 5ml. of trifluoroacetic acid the mixture was stirred at 1015 for 15minutes. The reaction mixture was poured into cold anhydrousether-Skellysolve B. After cooling for a short time the precipitate wascollected by filtration and washed with cold Skellysolve B-ether. Theproduct was stored in a vacuum desiccator over phosphorus pentoxide,yield 1.0 g. The product was combined with 40 ml. of water, 15 ml. of a25% solution of Amberlite LA-l resin acetate form in methylisobutylketone and 15 ml. of methyl isobutylketone and the mixturestirred for 1 hour. The phases were separated and the organic phase wasextracted twice with water. The extracts were combined with the aqueousphase and extracted 5 times with ether. The aqueous phase wasconcentrated to dryness and the residue triturated with 2-propanol. Theproduct was collected by filtration and washed with 2-propanol andseveral times with anhydrous ether; yield 0.52 g. The product was driedin vacuo over phosphorus pentoxide; M.P. decomposes above 185. Theinfrared and nuclear magnetic resonance spectra were consistent for thedesired compound.

Illustrative preparation of starting reagents (A) Ring substituted 2phenyl-glycines.Substituted DL 2-phenylglycines are prepared by alkalinehydrolysis of the appropriate 5-arylhydantoins which in turn areprepared from substituted benzaldehydes by the method of Buchnerer andLeib, J. Prakt. Chem., 141, 5 (1934); for additional examples of thisprocedure see also US. Pat. 3,140,282. The substituted DLZ-phenylglycines are resolved, if desired, via their N-formylderivatives as described by E. Fisher et al., Ber. 41, 1286 (1908) or byone of the procedures illustrated below.

D-ot-acetamido-phenylacetic acid.A suspension of 50 g. (0.331 mole) ofD-()2-phenylglycine in 700 ml. of water was cooled to 0 to 5 C. and 13.2g. (0.331 mole) of sodium hydroxide was added with stirring to produce asolution. Acetic anhydride (67.5 g., 0.662 mole) was added rapidly inone portion to the vigorously stirred solution which was initiallycooled to 0 to 5 C. by means of a salt-ice cooling bath. This wasimmediately followed by the addition of a solution of 39.7 g. (0.993mole) of sodium hydroxide in 200 ml. of water in a rapid stream from adropping funnel. The temperature rose to a maximum of about 25 C. Thesolution was stirred for an additional fifteen minutes in the coolingbath and then acidified with concentrated hydrochloric acid. Theprecipitated product was collected by filtration, washed on the filterwith water and recrystallized from 1:1 95% ethanol-water; yield 46.0 g.(72%), M.P. 186- 188 C., [a] 217.9 (C=1% ethanol).

D-u-acetamido 4 nitrophenylacetic acid.D-u-acetamido-phenylacetic acid(20 g., 0.104 mole) was slowly added to 50 ml. of concentrated sulfuricacid with cooling as needed to maintain the temperature at 20 to 25 C.The mixture was stirred for about 20 minutes until most of the soliddissolved. Nitric acid d.=1.5, 9.7 ml., 0.208 mole) was added dropwiseat such a rate to the stirred mixture that the salt-ice cooling bathmaintained the temperature in the range 0 to -5 C. The reaction mixturewas stirred at -5 to 10 C. for an additional 30 minutes and then pouredonto about 300 g. of ice flakes. The white crystalline product was 15collected by filtration, washed with water and recrystallized threetimes from 1:1 95% ethanol-water; M.P. 180-182 C. dec., yield 11.5 g.(46.4%). An additional recrystallization from ethyl acetate did notchange the melting point; [a] 206.4 (C==5%, ethanol).

Analysis.Calcd for C H N O (percent): C, 50.42; H, 4.23; N, 11.76. Found(percent): C, 50.14; H, 4.07; N, 11.96.

D-a-acetamido 4 aminophenylacetic acid.A solution of 15 g. (0.063 mole)of D-a-acetamido-4-nitrophenylacetic acid in 250 ml. of 95% ethanol washydrogenated in the presence of 0.6 g. of 5% palladium' on carbon on aPaar hydrogenator at an initial pressure of 50 p.s.i. for 64 minutes.The product had crystallized from the hydrogenation mixture.Approximately 200 ml. of water was added, the mixture warmed to dissolvethe product and the catalyst removed by filtration. Chilling thefiltrate gave 9.9 g. of product, M.P. 192-195 C. dec. The product wasrecrystallized four times from 1:1 95% ethanol-water, weight 4.8 g.,M.P. 207-209 C. dec., M1 -182.8 (C=0.5%, 1 N HCl).

Analysis.Calcd for C H N O (percent): C, 57.71; H, 5.81; N, 13.46. Found(percent): C, 57.61, 57.64; H, 5.67; N, 13.18.

D-a-acetamido 4 iodophenylacetic acid.To a solution of 5.0 g. (0.024mole) of D-u-acetamido-4-aminophenylacetic acid in 70 ml. oftrifluoroacetic acid at 5 to was added slowly 1.8 g. of sodium nitrite.The solution was stirred for 25 minutes. Solid potassium iodide (4.8 g.,0.024 mole) was added at 0- to The temperature of the dark brown mixturewas increased to 30 whereupon a vigorous gas evolution occurred. Themixture was maintained at 30 for 45 minutes and then heated at refluxfor one-half hour. The trifluoroacetic acid phase was decanted from thedark colored insoluble material. The trifiuoroacetic acid was distilledoff at reduced pressure. The residue was taken up in 50 ml. of water.After ice cooling there was obtained a precipitate of brown solid. Tworecrystallizations from 1:1 95% ethanol-water gaveD-a-acetamido-4-iodophenylacetic acid; M.P. 217 dec. with darkening at202, [a] 173.2 (C=0.5, 95% ethanol).

Analysis.-Calcd for C H INO (percent): C, 37.64; H, 3.16; N, 4.39. Found(percent): C, 37.84; H, 3.30; N, 4.34.

The dark colored insoluble material was slurried with water and treatedwith 1 M sodium thiosulfate to remove the iodine color. The solid wasfiltered, Washed with water and twice recrystallized from 1:195%ethanolwater with a carbon treatment giving additional product: [a]170.6 (C=0.5, 95% ethanol).

Analysis.Calcd for C H lNO (percent): C, 37.64; H, 3.16; N, 4.39. Found(percent): C, 37.70; H, 3.25; N, 4.45.

D-a-amino4-iodophenylacetie acid.A suspension of 3.7 g. (0.011 mole) ofD-aacetamido4-iodophenylacetic acid in ml. of 2 N hydrochloric acid plussuflicient dioxane to give a solution at the boiling point was heated atreflux for 1.5 hours. The solvent was distilled off at reduced pressure.The residue was extracted with water, the insoluble material (solid A)being removed by filtration. The filtrate was stripped to dryness atreduced pressure and the solid residue was extracted with water, theinsoluble material (solid B) again removed. The filtrate was againevaporated to dryness and the residue extracted with water and theinsoluble material (solid C) again removed. The filtrate was stripped todryness giving solid D as residue. The infrared spectra (KBr) showedsolids A and B to be amino acid zwitter ion and solid C to be mostlyamino acid hydrochloride.

Solid A was hydrolyzed in 2.5 N hydrochloric acid plus dioxane for 1.75hours. The solution was evaporated to dryness, the residue taken up inwater, a small amount of insoluble material removed by filtration, andthe filtrate 16 evaporated to dryness leaving solid E. An infrared spectrum (KBr) showed solid E to be a mixture of amino acid hydrochlorideand zwitter ion.

Solids D and B were combined in water and the system adjusted to pH 4.5giving 1.55 g. of D-a-amino-4-iodophenylacetic acid; M.P. 204-205" dec.,[a] 99.4 (C=0.5, 1 N HCl).

Analysis.-Calcd for C H INO (percent): C, 34.68; H, 2.91; N, 5.06. Found(percent): C, 34.63; H, 3.24; N, 4.77.

D-Ot-ElIIllI'lO 4-iodophenylacetic acid.D-a-acetamidophenylacetic acid(Beil. 14, 591) (200 g., 1.036 mole) was added slowly to a solution of161.1 g. (0.52 mole) of silver sulfate in 1.2 1. of cone. sulfuric acidwith cooling as needed to keep the temperature below 30. Finelypulverized iodine (684 g., 2.7 mole) was added in portions during 1.5hours. The mixture was stirred at room temperature for 1.5 hours longer.The mixture was filtered through a sintered glass filter and thefiltrate poured into ca. 3 l. of crushed ice. The solid was filtered,washed with water, and air dried. The material was recrystallized from650 ml. of 2-propanol (the hot solution was filtered to remove someinsoluble material) giving solid A; yield 46.6 g., M.P. 175183 dec. Thefiltrate was concentrated and stored in the cold overnight giving solidB; yield 132 g., M.P. 160168. Solids A and B were crudeD-ocacetamido-4iodophenylacetic acid.

Solid B was combined with 500 m1. of 2 N hydrochloric acid and refluxedfor one hour. The insoluble material (solid C) was removed by filtrationand washed with water; yield 100 g., M.P. ISO-183.

Solid C was hydrolyzed in 200ml. of 2 N hydrochloric acid plus enoughdioxane to solubilize the material. After 2.25 hours at reflux thesolvent was distilled off at reduced pressure and the residue extractedwith 250 ml. of water. The insoluble material (solid D) was removed byfiltration. The filtrate was adjusted to pH 4.5 and after cooling in anice bath the precipitate was filtered, washed with water, and trituratedwith boiling ethanol giving 10.8 g. of D-u-amino-4-iodophenylaceticacid.

Solid A was hydrolyzed in 2 N hydrochloric acid plus dioxane for twohours and the solvent distilled off at reduced pressure. The residue wasextracted with water. The insoluble material (solid F) was removed byfiltration. The filtrate was adjusted to pH 4.5 giving crystalline D-a-amino 4-iodophenylacetic acid; yield 14.4 g., [a] 86.2 (C-=0.5, 1 NHCl).

Solids D and F were combined and suspended in 350 ml. of water. Thesuspension was adjusted to pH 4.5 with 20% sodium hydroxide. The solidwas filtered, washed with water, air dried, and triturated with 300 ml.of boiling 95 ethanol giving 42.5 g. of D-a-arnino-4-iodophenylaceticacid; [a] 99.8 (C=0.5, l N HCl).

D-ot-amino 4 iodophenylacetyl chloride hydrochloride.A suspension of42.3 g. (0.15 mole) of finely ground D-oc-arnino 4-iodophenylacetic acidin 1.5 l. of methylene chloride was gassed at 0 to 5 with anhydroushydrogen chloride and 40.6 g. (0.195 mole) of phosphorus pentachlorideadded. The mixture Was stirred for two hours at 5 Skellysolve B (800ml.) was added to the reaction mixture and the product collected byfiltration. The product was washed with Skellysolve B and dried invacuo; yield 40.9 g. (82% D-a-acetamido-3iodophenylacetic acid.To asolution of 50.0 g. (0.24 mole) of Da-acetamido-3-aminophenyl aceticacid in 550 ml. of trifluoroacetic acid at 5 was added 17.0 g. of 97%sodium nitrite gradually during 10 minutes. The solution was stirred at5 for 25 minutes longer. The diazonium salt solution Was added in asteady stream to a vigorously stirred suspension of 79.0 g. of potassiumiodide and 2.5 g. of iodine in 300 ml. of tri fluoroacetic acidinitially at 23. During the addition the temperature rose to 2527 andsteady gas evolution was noted. The mixture was stirred for 3.5 hours atroom temperature until the gas evolution ceased. The reaction mixturewas filtered leaving a quantity of dark gummy solid. The filtrate wasconcentrated to a small volume, water added, and further concentrated.The concentrate containing a solid was treated with dilute sodiumthiosulfate and the solid collected by filtration. Recrystallizationfrom 4:1 water -95% ethanol gave 19.5 g. of D-oc-flCBtflHlldO-3-iodophenylacetic acid; M.P. 181181.5, [a] 158.0 (:0.5, 95% ethanol).

Analysis.Calcd. for C H INO (percent) C, 37.64; H, 3.16; N, 4.39; I,39.77. Found (percent): C, 37.80; H, 3.07; N, 4.55; I, 39.20.

D-a-amino-3-iodophenylacetic acid.D-a-acetarnido-3- iodophenylaceticacid (10.0 g.) in 45 ml. of 2 N hydrochrolic acid plus sufficientdioxane to give a solution at the boiling point was refluxed for 2.25hours. The solvent was evaporated to dryness and the residue extractedwith water. The insoluble material (solid A) was removed by filtration.The filtrate was evaporated to dryness and the residue in water wasadjusted to pH 4.5 with sodium hydroxide giving 1.0 g. ofD-a-arnino-3-iodophenylacetic acid; M.P. 192-195, [u] -81 (C, 0.5, 1NHCl).

The filtrate deposited a second crop of amino acid on storage in thecold; yield 0.6 g., M.P. 203204.5, [od 101.4 (C, 0.5, 1 N HCl).

Solid A (which was chiefly amide) was hydrolyzed with 45 ml. 2 Nhydrochloric acid plus dioxane for two hours at reflux. The residueremaining after evaporation of the solvent was combined with water andagain evaporated to dryness. The residue in water was adjusted to pH 4.5giving 1.9 g. of D-a-amino-3-iodophenylacetic acid; M.P. 196-199, [a]--95 (C, 0.5,1N HCl).

The infrared and nuclear magnetic resonance spectra of the threefractions Were consistent with the desired product.

D-a-amino-3-iodophenylacetyl chloride hydrochloride.A suspension of 2.5g. (0.009 mole) of D-a-amino- 3-iodophenylacetic acid in 100 ml. ofmethylene chloride at 0 to 5 was gassed with anhydrous hydrogen chlorideand 2.5 g. (0.012 mole) of phosphorus pentachloride added. Afterstirring for 24 hours at 0 to 5 an additional 1.2 g. of phosphoruspentachloride was added and stirring continued for a total of 38 hours.The reaction mixture was diluted with Skellysolve B, the productfiltered, washed with Skellysolve B, and dried in vacuo; yield 1.2 g.

D a-arnino-a-(3-chloro-4-hydroxyphenyl)glycine.--To a stirred suspensionof 5.01 g. (0.03 mole) of D-(-)-2-(p-hydroxyphenyl) glycine in 100 ml.of glacial acetic acid was bubbled in HCl gas at a vigorous rate forabout 5 minutes. At first a clear solution resulted and then thehydrochloride salt crystallized out. Next, 4.45 g. (0.033 mole) ofsulfuryl chloride (freshly distilled) in 25 ml. of glacial acetic acidwas added, with stirring, over a 30 minute period, dropwise. Thetemperature was 26- 27 C. throughout the addition. After one hourstirring, 250 ml. of dry ether was added slowly and crystallizationbegan. After min. the product was filtered off, washed with dry etherand air dried. The 7 g. obtained was dissolved in 50 ml. of 1 N HCl,filtered, and the pH adjusted, with cooling to 5 with cone. NH OH. Theresulting crystalline product was filtered off after 5 min. standing,washed with two ml. portions of water and 5 with acetone. The vacuumdried material weighed 4.6 g.; dec. pt. 217 C. (sharp). The NMR and IRspectra were consistent with the desired structure. [a] C. 137.1 (C,1%,1NHC1) Analysis.-Calcd for C H CINO (percent): C, 47.76; H, 4.01; Cl,17.66. Found (percent): C, 47.16; H, 3.92; CI, 17.96.

dI-Z-(p-methoxyphenyl)-glycine.To a stirred solution of 19.6 g. (0.4mole) of NaCN in 80 ml. of H 0 was added 23.6 g. (0.450 mole) of NH CIand 20 Ml. of conc. NH OH followed by 54.5 g. (0.4 mole) of anisalde--hyde in 160 ml. of methanol and the temperature maintained at 37 C. fortwo hours. The methanol was then removed in vacuo and the remainingmixture extracted with two ml. portions of methyl isobutyl ketone (MIBK)and combined. The combined MIBK extracts were washed once with 30 ml. ofH 0 and then 240 ml. of 6 N HCl added with good mixing and the MIBK wasremoved in vacuo. The resulting slurry was heated at re flux (now insolution) for two hours. One hundred ml. of H 0 was added to the hotsolution and then 8 g. of decolorizing carbon added and after tenminutes at gentle reflux the carbon was filtered off and washed with 50ml. of hot water. The combined filtrates (hot) were stirred and treatedwith conc. NH OH until pH 5-6 was obtained (pH paper). The slurry wasthen cooled to 5 C. and after one hour the crystals were filtered offand washed with two 100 ml. portions of Water. The damp cake was thenslurried in 250 ml. of water and 50% NaOH added slowly until the productdissolved. Two 300 ml. ether extracts were then taken and discarded. ThepH was then adjusted to 5.5 with 6 N HCl with cooling. After one hourthe product was filtered off, washed with 3x 100 ml. H 0 and air dried.Yield 40 g.; dec. 244 C. with sublimation at 230 C.

dl Z-(p-methoxyphenyl)-N-(chloroacetyl)-glycine.- To a stirredsuspension of 36 g. (0.2 mole) of dl-Z-(pmethoxyphenyl)-glycine in 500ml. of H 0 was added 8 g. (0.2 mole) of NaOH pellets and then a clearsolution was obtained the solution was cooled to 5 C. and with vigorousstirring 68.2 g. (0.4 mole) of chloroacetic anhydride (warm) was addedall at once. Then a solution of 16 g. (0.4 mole) of NaOH in 100 ml. of H0 was added over a 10 to 15 minute period. More 20% NaOH was added asneeded to keep the pH at about 9 for a. 1.5 hour period. Next, the pHwas adjusted to 2 with 40% H PO The product crystallized immediately andwas filtered off, washed with water and recrystallized fromethanol-water to give 38 g. of product melting at 182- 183 C.

Analysis.Calcd for C H CINO (percent): C, 51.21; H, 4.69. Found(percent): C, 51.49; H, 4.90.

D 2 (p-methoxyphenyl)-N-chloroacetylglycine andL-(+)-2-(p-methoxyphenyl)-glycine.-To 800 m1. of H 0 stirred at 37 'C.was added 38 g. (0.148 mole) ofdI-Z-(p-methoxyphenyl)-N-chloroacetylglycine and added dropwise until pH7.8 was obtained. To the resulting solution was added 2 g. of hog kidneyacylase (Sigma Chemical Company) and stirring continued at 37 C.(internal) for 21 hours. The solids containing crude L-(-|-)-2-(p-methoxyphenyl)-glycine were then filtered off and washed with 2x100ml. H 0 and the pH of the combined filtrates adjusted to 45 with glacialacetic acid. This solution was heated on the steam bath for 30 min. with5 g. of decolorizing carbon and then filtered. The carbon cake wasWashed with 50 ml. of warm water and the combined filtrates cooled andacidified to pH 2 with 40% H PO After one hour cooling at 0 C. thecrystalline product was filtered oif and washed with cold water (3 andair dried. The yield was 16 g.D-()-2-(pmethoxyphenyl)-N-chloroacetylglycine and when a second runusing 5x the above amounts were used a yield of 83 g. (87% yield) wasobtained. M.P. -171 C.; [u] 193 (C, 1%, ethanol).

Analysis.--Calcd for C H ClNO (percent): C, 51.21; H, 4.69. Found(percent): C, 51.50; H, 4.99.

When the solids containing crude L-(+)-2- (p-methoxyphenyl)-glycine aretreated with hot 3 N HCl (200 ml.) and carbon followed by filtration andpH adjustment to 5.5 there is obtained 6 g. (first run) of pureL(+)-2-(pmethoxyphenyl)g1ycine. [u] +150.4 (C=1%, 1 N HCl).

D-()-2-(p-methoxyphenyl)-glycine.The 16 g. of D ()-2-(p-methoxyphenyl)-N-chloroacetylglycine was refluxed 1.5 hours in 170 ml. of 2 N HCl. Theresulting clear solution was filtered and cooled at 5 C. and the pHadjusted to 5.5 with NH OH.

19 The product was then filtered off after cooling 30 min. and washedwith 3 X 25 ml. of cold water. The dried materialD-(-)-2-(p-methoxyphenyl) glycine weighed 9.5 g. A second run gave 54 g.

[a] 149.9 (C=1%, 1 N HCl) (first run) [u] 148.1 (C=1%, 1 N HCl) (secondrun) Analysis.Calcd for C H =NO (percent): C, 59.67; H, 6.13; N, 7.74.Found (percent): C, 59.38; H, 6.16; N, 8.00.

D-(-)-Z-(p-hydroxyphenyD-glycine.-A mixture of 1.81 g. (0.01 mole) ofD-()-2-(p-methoxyphenyl)glycine ([a] 149.9 C=1%, 1 N HCl) and 10 ml. of48% HBr was heated at gentle reflux for 2 hours. The resulting solutionwas concentrated at reduced pressure at 30 C. to a wet solid. A minimumamount of water (20 C.) was added to dissolve the HBr salt and withcooling NH OH was added to pH 5. The resulting thick gel which ppt. waswarmed to 50 C. and when solution was nearly obtained a differentcrystalline form began to ppt. Upon cooling 30 min. at -5 C. there wasobtained 990 mg. of cold water washed (3X 1 ml.) and air dried material,D-()-2-(p-hydroxyphenyDglycine. [a] 161.2 (C=1%, 1 N HCl) dec. pt. 223C. A second run using 20X the above amounts gave 24.5 g. of material.[a] 153 (C=1%, 1 N HCl).

Analysis.Calcd for C H NO (percent): C, 57.49; H, 5.43; N, 8.39. Found(percent): C, 57.41; H, 5.67; N, 8.39.

-D 2-(3,5-dichloro-4-hydroxyphenyl)-glycine. To a stirred suspension of5.01 g. (0.03 mole) of D-( )-2- (4-hydroxyphenyl)glycine in 100 ml. ofglacial acetic acid was bubbled in HCl gas at a vigorous rate for about5 minutes. At first a clear solution resulted and then the hydrochloridesalt crystallized out. Next, 9.0 g. (0.067 mole) of sulfuryl chloride(freshly distilled) in 25 ml. of glacial acetic acid was added, withstirring, over a 30 minute period, dropwise. The temperature was 26-27C. throughout the addition After the sulfuryl chloride addition, theslurry was heated to 70 C. for 30 minutes and then stirred at ambienttemperature for two hours. Then 250 ml. of dry ether was added slowlyand crystallization began. After min. the product was filtered off,washed with dry ether and air dried. The 7 g. obtained was dissolved in100 ml. of 1 N HCl, filtered, and the pH adjusted, with cooling to 5with cone. NH OH. The resulting crystalline product was filtered offafter 5 min. standing, washed with two ml. portions of water and 5X withacetone. The vacuum dried material Weighed 4.5 g.; dec. pt. 210 C.(sharp). The NMR and IR spectra were consistent with the desiredstructure. [a] -126.3 (C=1%,1-N HCl).

Analysis.Calcd for C H Cl NO (percent) C, 40.78; H, 2.99; Cl, 30.04.Found (percent): C, 41.85; H, 3.22; Cl, 27.80.

Resolution of DL oz amino 3 methoxyphenylacetic acid.DL 0camino-3-methoxyphenylacetic acid [A. H. Neims, D. C. De Luca, L.Hellerman, Biochemistry, 5 (1), 203 (1966)] was resolved withd-IO-camphorsulfonic acid in water.

DL-kx-amino-3-methoxyphenylacetic acid (331 g., 0.182 mole) was added toa solution of 46.4 g. (0.2 mole) of d-10-camphorsulfonic acid in 135 ml.of water at 50 to 60. The solution was filtered and stored in the coldfor 20 hours. The precipitated amino acid d-IO-camphorsulfonate salt Wascollected by filtration. The salt was repeatedly recrystallized fromwater until a sample of the amino acid regenerated from it showed nofurther change in optical rotation. Thus, after three recrystallizationsfrom water, there was obtained 3.7 g. of the d-lO-carnphorsulfonate saltof D-a-amino-3-methoxyphenylacetic acid; M.P. 184-185 dec. The salt (1.4 g.) was dissolved in about 10 ml. of water by warming. The solutionwas adjusted to pH 5-6 with concentrated ammonium hydroxide. The productwas allowed to crystallize first at room temperature and then in an icebath giving, after filtration and drying in vacuo over phosphoruspentoxide. 0.36 g. of D-u-amino-3-methoxyphenylacetic acid; M.P. 178181dec., [a] 129.0 (C=O.5, 1 N HCl). A portion of the amino acid wasrecrystallized from water and dried in vacuo over phosphorus pentoxide;M.P. 180182 dec., [a] -136 (C=0.08, 1 N HCl).

Analysis.-Calcd for C H NO /3H O (percent): C, 57.74; H, 6.28; N, 7.48.Found (percent): C, 57.70, 57.76; H, 6.23, 6.18; N, 7.21.

D ot-amino-3-hydroxyphenylacetic acid hydrobromide monohydrate.-D 0camino-3-methoxyphenylacetic acid (2.9 g., 0.016 mole) and 16 m1. of 48%hydrobromic acid were refluxed for two hours. The volatile materialswere removed at reduced pressure. Water (about 15 ml.) was added to theresidue and this removed at reduced pressure. This was repeated once.The residue was dried in vacuo to remove all water. The dried residuewas recrystallized by dissolving in 2-propanol and adding Skellysolve Bto the cloud point. After drying there was obtained 3.0 g. of D a amino3 hydroxyphenylacetic acid hydrobromide monohydrate; M.P. 156-162" dec.,[a] --62 (C=0.1, water). The infrared and nuclear magnetic resonancespectra were consistent with the desired product.

Analysis.-Calcd for C H NO -HBr-H O (percent): C, 36.10; H, 4.55; N,5.26. Found (percent): C, 37.03; H, 5.12; N, 5.34.

(2) D-a-acetamido-3-nitrophenylaceti'c acid.-A stirred suspension of49.8 g. (0.254 mole) of D-a-arnino-3-nitrophenylacetic acid [P. Fries,K. Kjaer, Acta Chimica 'Scand., 17, 2391 (1963)] in 500 ml. of water wascooled in an ice bath and a solution of 8.36 g. (0.209 mole) of NaOH in40 ml. of water was added causing most of the solid to dissolve. Therewas immediately added 42.7 g. (0.418 mole) of acetic anhydride followedby the addition as needed of a solution of 25.1 g. (0.627 mole) ofsodium hydroxide to maintain the pH value at about 7. The reactionmixture was stirred in the ice bath for an additional 15 minutes,filtered, and adjusted to pH 1.8 with concentrated hydrochloric acid.The crystalline product was collected by filtration and washed withwater; yield 25 g., M.P. 172174 dec. The product was twicerecrystallized from 1:1 ethanol-water giving, after drying in vacuo overphosphorus pentoxide, 11.8 g. of D-a-acetamidO-3- nitrophenylaceticacid, M.P. l83185; [a] 179.4 (0:05, 95% ethanol). The infrared andnuclear magnetic resonance spectra were consistent with the desiredcompound.

Analysis.Calcd for C H N O (percent): C, 50.42; E11, $.23; N, 11.76.Found (percent): C, 50.56; H, 4.20; N,

D-a-acetamido-3-aminophenylacetic acid.A solution of 9 g. (0.03 78 mole)of D-a-acetamido-3-nitrophenylacetic acid in ml. of methanol washydrogenated using 0.6 g. of 5% palladium on carbon at an initialpressure of 50 psi. on a Paar hydrogenation apparatus for 30 minutes.The hydrogenation bottle was cooled with a jet of air to keep thetemperature under 40". The catalyst was removed by filtration.Evaporation of the filtrate gave a crystalline product. Tworecrystallizations from l-propanol gave 3.4 g. ofD-a-acetamido-3-aminophenylacetic acid, M.P. 200 201 dec.; [06113 174.4(C=0.5, water).

Analysis.'Calcd for C I I N O (percent): C, 57.71; II-IB, 35581; N,13.46. Found (percent): C, 57.78; H, 5.97; N,D-a-amino-3-hydroxyphenylacetic acid.A solution of 2.1 g. (0.01 mole) ofD-a-acetamido3-aminopheny1acetic acid in 35 ml. of trifluoroacetic acidwas cooled to 5 and 0.69 g. (0.01 mole) of solid sodium nitrite added.After stirring for 20 minutes at 5 acetic acid (5 ml.) was added. Themixture was stirred at 45 to 50 for one and one-half hours and thenheated on the steam bath for one-half hour. The cold reaction mixturewas poured onto 30 g. of crushed ice. The volatile material-s weredistilled at reduced pressure leaving as residue at slightly brownviscous oil. The residue was combined with 30 ml. of 2 N hydrochloricacid and refluxed for one and one-half hours. The volatile materialswere removed under reduced pressure. Water was added to the residue andthis removed under reduced pressure causing the hydrochloride salt ofthe product to crystallize. The residue was dissolved in a minimumamount of water, adjusted to pH 4.5 with 20% sodium hydroxide, filtered,and stored in the cold giving 0.43 g. of crystallineD-or-amino-3-hydroxyphenylacetic acid, M.P. 204-206 dec. The filtratewas stripped to dryness and a small amount of water added to thecrystalline residue giving a second crop (0.46 g.) of the amino acid.

The filtrate from the 2nd crop plus 3 ml. of concentrated hydrochloricacid were concentrated to dryness. The cooling gave crystallineD-a-amino-3-hydroxyphenylacetic acid hydrochloride monohydrate; yield0.5 g., M.P. ISO-153 dec., [u] -91.2 (C=0.5, water). The infrared andnuclear magnetic resonance spectra were consistent with the desiredproduct.

Analysis.--Calcd for C H NO- -HCl-H O (percent): C, 43.35; H, 5.46; N,6.32. Found (percent): C, 42.7; H, 5.6; N, 6.17; residue, 1.45. Valuescorrected for 1.45% residue: C, 43.3; H, 5.7; N, 6.26.

The two crops of D-u-amino-3-hydroxyphenylacetic acid were combined,suspended in a small amount of water, 2 ml. of 48% hydrobromic acidadded and the filtered solution evaporated to dryness. The residue wastwice recrystallized from water giving 150 mg. of D-u-amino-3-hydroxyphenylacetic acid hydrobromide monohydrate; M.P. 172175 dec.,[orb -74 (C=0.1, water). The infrared and nuclear magnetic resonancespectra were consistent with the assigned structure.

Analysis.-Calcd for C H NO -HBr-H O (percent): C, 36.10; H, 4.55; N,5.26. Found (percent): C, 36.20; H, 4.62; N, 5.32.

D-a-amino-3-hydroxyphenylacetic acid.A solution of 38.4 g. (0.1845 mole)of D-a-acetamido-3-aminophenylacetic acid in 600 ml. of trifluoroaceticacid prepared at 15 to 20 Was cooled to and 13 g. (0.1845 mole) of 98%sodium nitrite added in portions during a -minute period with stirringat 5 After stirring for an additional 25 minutes 90 ml. of acetic acidwas added at 5 to 0. The mixture was heated at 45 to 50 for one andone-half hours (gas evolution) refluxed for one-half hour, cooled, andpoured onto 500 g. of ice flakes. The volatile materials were removed atreduced pressure. The residue was refluxed With 400 ml. of 2 Nhydrochloric acid for one hour. Concentration to a small volume gave thecrystalline hydrochloride salt. The dried product (27 g.) wasrecrystallized from wet acetic acid (150 ml. acetic acid plus 7 ml. ofwater) giving 21 g. of D-ot-amino-3-hydroxyphenylacetic acidhydrochloride monohydrate; M.P. 149152 dec., [a] 103.0 (C=0.5, water).

D a-arnino-4-acetamidophenylacetic acid.D-a-acetamido4-aminophenylacetic acid (39.2 g., 0.188 mole) in 400 ml. of 2 Nhydrochloric acid Was refluxed for two hours. The mixture wasconcentrated to dryness at reduced pressure. Water was added and thesolution again concentrated to dryness. This was repeated once. Thecrystalline residue was slurried with 2-propanol, filtered, and washedadditionally with 2-propanol giving, after air drying, 47 g. of thehydrochloride of D-u-amino-4-aminophenylacetic acid.

Ten g. of the hydrochloride in 40 ml. of water was adjusted to pH 4.8with 20% sodium hydroxide. Crystalline D-u-amino-4-aminophenylaceticacid separated. To the solution obtained by adding 160 ml. additional ofwater was added 10 ml, of thioacetic acid. The mixture was stirred for17 hours at 24 under a nitrogen atmosphere. The reaction mixture,containing a quantity of crystalline product, was concentrated toone-half of its initial volume giving 4.5 g. of product. The crudeproduct was suspended in water, the suspension adjusted to pH 4.6 with20% NaOH, heated to 95, carbon treated, and the product allowed tocrystallize in the cold overnight. The resulting gelatinous mass wasbroken up by warming. The solid was removed by filtration, Wt. 0.2 g.,M.P. 203206 dec. The filtrate was diluted with an equal volume ofethanol giving 1.4 g. of D-wamino-4-acetamidophenylacetic acid; M.P.214215 dec., [u] 133.4 (0:05, 1 N HCl).

Analysis.-Calcd for C H N O (percent): C, 57.71; H, 5.814; N, 13.46.Found (percent): C, 56.80, 56.72; H, 5.84, 5.89; N, 13.62; H O, 1.32.Found values corrected for 1.32% water: C, 57.52; H, 5.71; N, 13.80.

D-a-arnino-3-aminophenylacetic acid.A solution of 9.8 g. (0.05 mole) ofD-a-amino-3-nitrophenylacetic acid [P. Friis and A. Kjaer, Acta, ChimicaScand. 17, 2391 (1963); British patent specification, 1,033,257] in 200ml. of water was prepared by adjusting the mixture to pH 9.3 withconcentrated ammonium hydroxide. The solution was hydrogenated for 1hour in the presence of 0.4 g. of 5% palladium on carbon on a Paarhydrogenation apparatus at an initial pressure of 50 p.s.i. The vesselwas cooled as needed to keep the temperature from going above 30. After1 hour an additional 0.4 g. of catalyst was added and hydrogenationcontinued for 1 hour longer. Three additional runs were madehydrogenating a total of 39.4 g. of nitro compound. Addition of thesecond amount of catalyst was omitted in the additional runs and ahydrogenation time of about 1 hour was used. Each run was filtered toremove catalyst, the filtrates pooled and concentrated to a small volumeuntil crystallization of the product started. The concentrate wasdiluted with about five volumes of 95 ethanol, the mixture storedovernight in the cold and the product filtered and washed further byslurrying with 95 ethanol, After drying in a vacuum oven for 3 hours at40 and then in vacuo over phosphorus pentoxide for 64 hours there wasobtained 25.7 g. of D-uamino-3-aminophenylacetic acid; M.P. 188-191",[a] 139.0 (6:1, in HCl).

The preparation of this compound has been described lgygP. )Friis and A.Kjaer, Acta Chimica Scand. 17, 2391 D-u-amino-3-acetamidophenylaceticacid.A mixture of 5 g. (0.0301 mole) of D-a-amino-3-aminophenylaceticacid and 5 ml. of thioacetic acid in ml. of water was stirred for 16hours under a nitrogen atmosphere. The mixture was heated on a steambath for one-half hour and then concentrated at reduced pressure to asmall volume. On cooling the concentrate the product started tocrystallize. The concentrate was diluted with 95% ethanol and, afterchilling in an ice bath, the product was filtered and Washed with 95ethanol; wt. 1.8 g. The filtrate was further diluted with 95 ethanolgiving an additional 2.3 g. of product. The two crops of product werecombined, dissolved in a small amount of water by warming, the solutionconcentrated slightly and diluted with a large volume of 95% ethanol.The initial crop of solid was removed by filtration and the filtratestored in the cold for 16 hours giving, after drying at 65 for 3 hoursin vacuo over phosphorus pentoxide, 0.90 g. of product, M.P. 185-187dec. The product was twice recrystallized from 1:1 95 ethanol-water: wt.0.36 g., M.P 186-187 dec., [a] (0:05, 1 N HCl).

Analysis.Calcd for C H N O (percent): C, 57.7; H, 5.81; N, 13.5. Found(percent): C, 47.29; H, 6.79; H, 11.21; H O, 18.3. Found valuescorrected for 18.3% Water: C, 57.9; H, 5.83; N, 13.7.

DL-u-amino-3-chl0rophenylacetic acid.A solution of 250 g. ofm-chlorobenzaldehyde in 1.5 l. of 95 ethanol was added in one portion toa stirred solution of 123 g. of sodium cyanide, 515 g. of ammoniumcarbonate and 1.5 1. of water. The mixture was stirred at 50 for 120hours. The cooled reaction mixture was acidified to pH 2 withconcentrated hydrochloric acid and stirred one hour. The hydantoin wascollected by 23 filtration, washed with cold water, and sucked dry onthe filter.

A mixture of the crude hydantoin obtained from two runs and 4 1. of 10%sodium hydroxide was refluxed for 18 hours. The solution was carbontreated and neutralized to pH 7 with acetic acid. The solid wascollected by filtration, washed with water, and dried and the filter. Asuspension of the product in 4 1. of water was acidified to pH 2 withconcentrated hydrochloric acid. After stirring for 1.5 hours theinsoluble material was removed by filtration and the filtrate adjustedto pH 7 with 10% sodium hydroxide. The precipitate was collected byfiltration and dried in vacuo at 75 for 18 hours giving 311 g. ofDL-ot-amino-3-chlorophenylacetic acid; M.P. 266269 dec.

DL-a-formamid-3-chlorophenylacetic acid.To 100 g. ofDL-a-amino-3-chlorophenylacetic acid was added 1.33 1. of formic acid.The reaction mixture was warmed to 50 and 483 ml, of acetic anhydridewas added dropwise. After storage overnight the DL-a-formamido-3-chlorophenylacetic acid was collected by filtration and washed withwater; yield 97 g.

D-(-)-u-amino 3 chlorophenylaceticacid.ot-Formamido-3-chlorophenylacetic acid (721 g.) and one kg. ofdehydroabietylamine were combined in 4.1 of methanol. After storing inthe cold for two hours the crystalline salt was collected by filtration.The product was recrystallized from methanol-water; yield 598 g., [u]--225 (0:0.4, methanol), The salt was slurried in 2 1. of methanol and 2liters of saturated sodium bicarbonate solution. The mixture was dilutedwith 2 1. of water, layered with methyl isobutyl 'ketone, and stirredvigorously. The aqueous phase was separated and acidified to pH 2 withcone. hydrochloric acid. The acid was collected by filtration and dried.The dried product was combined with 2 1. of 6 N hydrochloric acid and750 ml. of methanol, the mixture heated for two hours. and filtered. Thesolution was adjusted to pH with ammonium hydroxide. The solid wascollected by filtration and washed with water and acetone giving 112 g.of D-()-ot-amino-3-chlorophenylacetic acid; [a] 125 (0:0.4, 1 N HCl).

D-()-u-amino3-chlorophenylacetyl chloride hydrochloride-To a stirredsuspension of 25 g. of D-(-)-oamino-3-chlorophenylacetic acid in 375 ml.of methylene chloride at 2 was aded 36.5 g. of phosphorus pentachloride.After stirring at 0 to 2 for one and one-half hours the productD-()-a-amino3-chlorophenylacetyl chloride hydrochloride was collected byfiltration, washed with methylene chloride and Skellysolve B, and driedin vacuo to constant weight; yield 17.0 g.

DL-ot-amino-3 fiuorophenylacetic acid.-To a stirred solution of 24.5 g.of sodium cyanide, 29.5 g. of ammonium chloride, 25 ml. of ammoniumhydroxide and 100 ml. of water at room temperature was added a solutionof 62.0 g. of m-fiuoro-ben'zaldehyde in 200 ml. of methanol. The mixturewas stirred at 38 for two hours. The methanol was stripped off atreduced pressure. The residue was extracted with two by 200-ml. portionsof ethyl acetate. The combined extracts were washed with water. To theethyl acetate phase was added dropwise with vigorous stirring 50 ml. of6 N hydrochloric acid at room temperature. The solution was put undervacuum (water aspirator) and 250 ml. of 6 N hydrochloric acid addeddropwise. The mixture was refluxed for 2.5 hours, stirred for 13 hours,and adjusted to pH 4.8 with concentrated ammonium hydroxide whilecooling in an ice bath. The aqueous phase was decanted and the gummyprecipitate triturated with water and ethyl acetate. The productDL-a-amino-3-fluorophenylacetic acid was collected by filtration anddried in vacuo over phosphorus pentoxide; yield 8.6 g., M.P. 200203(sublimation). A second crop of product separated from the filtrates;yield 2.0 g., M.P. 245250 (sublimation).

DL-u-formamido-3-fiuorophenylacetic acid.--A partial solution of g. ofDL-ot-amino-3-fluorophenylacetic acid in 356 ml. of 88% formic acid washeated to and 119 ml. of acetic anhydride added dropwise. The mixturewas stirred for 17 hours at 5060 and cooled. The product,DL-a-formamido-3-fluorophenylacetic acid, was collected by filtrationand dried in vacuo; yield 38.5 g., M.P. 207-209" dec.

D-a-formamido-3-fiuorophenylacetic acid.-To a solution of 20 g. ofDL-ot-formamido-3-fluorophenylacetic acid in 4 l. of pH 7 phosphatebuffer was added 3.0 g. of hog kidney D-amino acid oxidase (NutritionalBiochemicals Corp.). The mixture was stored at 37 for 19.5 hours,adjusted to pH 5.0 with acetic acid, 5 g. of carbon added, heated to forone-half hour and filtered. The filtrate was adjusted to pH 2 with 40%phosphoric acid and extracted with ethyl acetate. The ethyl acetateextract was washed with water and stripped to dryness giving 10.0 g. ofproduct; M.P. 190-192", [a] 161.0 (C=l.0, methanol). The product wasagain treated with hog kidney D-amino acid oxidase (1 g.) in 500 ml. ofpH 7 phosphate buffer and the product worked up as above. There wasobtained after recrystallization of the product from methanol 5.7 g. ofD-a-formamido-3-fluorophenylacetic acid; [a] -178.0 (C=1.0, methanol).

D-a-amino-3-fluorophenylacetic acid.--A suspension ofD-a-formamido-3-fiuorophenylacetic acid (9.48 g.) in ml. of 6 Nhydrochloric acid was refluxed for onehalf hour. The reaction mixturewas cooled in an ice bath, filtered, and adjusted to pH 3.8 withconcentrated ammonium hydroxide. After stirring for 10 minutes theproduct, D-u-amino-3-fluorophenylacetic acid, was collected byfiltration and dried in vacuo over phosphorus pentoxide; yield 6.63 g.,[a] (C=l.0, 1 N hydrochloric acid), M.P. 249250.

Additional information is given concerning the synthesis ofring-substituted 2-phenylglycines by Doyle et al., J. Chem. Soc., 1440(1962) and Ryan et al., J. Med. Chem., 12, 310-313 (1969). As stated byRyan et al. such amino acids are converted into N-t-butoxycarbonylderivatives by the method of Schwyzer et al., Helv. Chim. Acta, 42, 2622(1959); Ryan et al., also gives an illustrative example of theresolution of such a derivative by the use of cinchonine.

(B) Ring-substituted cephaloglycins with and without blocked ot-aminogroups D( )-a( 3 chloro 4hydroxyphenyl)-a-(t-butoxycarboxylamino)-acetic acid.-To a stirredsuspension of 4.0 g. (0.02 mole) of D-(-) 2 (3-chloro-4-hydroxyphenyl)glycine (finely ground) and 1.6 g. (0.04 mole) of powdered magnesiumoxide in 50 ml. of 1:1 dioxanewater was added 5.8 g. (0.04 mole) oft-butoxycarbonylazide (Aldrich Chemical Company Inc.) over a 30 minuteperiod and then stirring continued for 20 hours at 45 50 C. Theresulting turbid solution was then poured into one liter of ice waterwith stirring. One 600 ml. ethyl acetate extract was taken and this waswashed twice with 200- ml. portions of 5% sodium bicarbonate and theseaqueous solutions combined and filtered. Next, with cooling, they wereacidified to pH 3 with 40% phosphoric acid under a layer of 500 ml. ofethyl acetate. This ethyl acetate extract was separated and combinedwith two more 100 ml. ethyl acetate extracts and dried over sodiumsulfate. The ethyl acetate solution was then filtered and concentratedunder reduced pressure to an oil and 100 ml. of warm benzene added. Theresulting solution was filtered. After removing the solvent in vacuothere was obtained 6 g. as an amorphous froth of D-()-a-(3 chloro4-hydroxyphenyl)-a-(t-butoxycarbonylamino)-acetic acid. Infrared and NMRanalysis revealed only the NH group had reacted with the azide.

7 [D-u-(t-butoxycarbonylamino)a(3chloro-4-hydroxyphenyl)-acetamido]cephalosporanic acid.To a stirredsolution of 4.03 g. (0.02 mole) of D-(-)-u-(3- chloro 4hydroxyphenyl)-ot-(t-butoxycarbonylamino)- acetic acid, 2.8 ml. oftriethylamine (0.02 mole) in 100 ml. of tetrahydrofuran, was added 3.64g. (0.02 mole) of trichloroacetyl chloride in 25 ml. of tetrahydrofuranover a 10 min. period at 40 C. (internal). After an additional 10 min.at 40 C., a pre-cooled solution at 50 C. of 5.44 g. (0.02 mole) of7-ACA, 5.6 ml. (0.04 mole) of triethylamine in 300 ml. of CH Cl wasadded all at once. The temperature was maintained at -40 C. to 30 C. for30 min. and then the cooling bath was removed and after another 30 min.(T max. C.) the solvent was removed in vacuo at 20 C. The residue wastaken up in 150 ml. of H 0 and 150 ml. of ether. The ether layer wasdiscarded and the aqueous layered with 150 ml. of ethyl acetate andcooled and stirred while being acidified to pH 2.5. The ethyl acetateextract was washed with water, dried 10 min. over N21 SO filtered andcone. to an oil at 20 C. under reduced pressure. The oil was triturateduntil a solid ppt. with two 200 ml. portions of 1:1 by volume dryether-Skellysolve B (pet. ether). The solids were filtered off andvacuum dried over P 0 The yield was 7.4 g. dec. pt. 100 C., slowly. TheIR and NMR were consistent with the desired structure.

Analysis.Calcd for 'C H ClN O S (percent): C,

, 49.64; H, 4.71. Found (percent): C, 49.50; H, 5.48.

7 [D a amino or (3 chloro 4 hydroxyphenyl)- acetamido] cephalosporanicacid.-A solution of 7 g. of 7 [D oz (t butoxycarbonylarnino) a (3chloro- 4 hydroxyphenyl) acetamido] cephalosporanic acid in 200 ml. of50% aqueous formic acid was stirred and heated at 40 C. for 3 hours andthen the solvents removed in vacuo at 20 C. to leave a glass-likeresidue. This was further dried by adding 200 ml. of toluene andremoving same under reduced pressure at 20 C. The residue was stirredwith moist ethyl acetate until solid and the solids filtered off. Next,the solids were stirred with 95% ethanol (200 ml.) for one hour andfiltered. There was obtained 2.5 g. of vacuum dried material. This wasfurther purified and crystallized by stirring for 2 hours in a mixtureof 12 ml. H 0 and 12 ml. of Amberlite LA-1 resin (acetate form) 25% inmethyl isobutyl ketone (MIBK). The product was filtered ofi, washed witha little MIBK-H O (1:1) and finally washed with acetone. The final yieldwas 450 mg. dec. 100 C. slowly. The IR and NMR were consistent with thedesired structure.

Analysis.'Calcd. for C H ClN O S (percent): C, 47.37; H, 3.94. Found(percent): C, 47.33; H, 4.98.

Am'berlite LA-l is a high molecular weight, waterinsoluble, liquidsecondary amine, commercially available from Rohm and Haas Co. Theacetate form used in this investigation was prepared as follows. To 1.0l. of Amberlite LA-l and 3.0 l. of isobutyl methyl ketone was added 120ml. of glacial acetic acid and the solution was stirred for 5 min. Afterstirring with 800 ml. of water for 25 min. the organic layer wasseparated for use.

More precisely, Amberlite LA-l is a mixture of secondary amines whereineach secondary amine has the formula wherein each of R R and R is analiphatic hydrocarbon radical and wherein R R and R contain in theaggregate from 11 to 14 carbon atoms; this particular mixture ofsecondary amines, which is sometimes referred to in the followingexamples as Liquid Amine Mixture No. I, is a clear amber liquid havingthe following physical characteristics: viscosity at 25 C. of 70 cps.;specific gravity at 20 C. of 0.845; refractive index at 25 C. of 1.467;distillation range at mm.: up to 160 C., 4%, 160 to 210 C., 5%, 210 to220 C., 74%, above 220 C., 17%.

D a (p hydroxyphenyl) or (t butoxycarbonylamino)acetic acid.To a stirredsuspension of 8.35 g. (0.05 mole) of D 2 (p hydroxyphenyl) glycine(finely ground) and 8 g. (0.2 mole) of powdered magnesium oxide in 225ml. of 1:1 dioxane-water was added 14.3 g. (0.10 mole) oft-butoxycarbonylazide (Aldrich Chemical Company Inc.) over a 30 minuteperiod and then stirring continued for 20 hours at 4550 C. The resultingturbid solution was then poured into one liter of ice water withstirring. One 600 ml. ethyl acetate extract was taken and this waswashed twice with 200 ml. portions of 5% sodium bicarbonate and theseaqueous solutions combined and filtered. Next, with cooling, they Wereacidified to pH 3 with 40% phosphoric acid under a layer of 500 ml. ofethyl acetate. This ethyl acetate extract was separated and combinedwith two more 100 ml. ethyl acetate extracts and dried over sodiumsulfate. The ethyl acetate solution was then filtered and concentratedunder reduced pressure to an oil and 100- ml. of warm benzene added. Theresulting solution was filtered and scratched. There was obtained 10.8g. of crystalline material, D a (p hydroxyphenyl)-u-(t-butoxycarbonylamino)acetic acid. Infrared and NMR analysis revealedonly the NH group had reacted with the azide.

Analysis.Calcd for C13H17NO5 (percent): C, 58.43; H, 6.48; N, 5.25.Found (percent): C, 62.46; H, 6.55; N, 4.56.

7 [D oz (t butoxycarbonylamino) oz (phydroxyphenyl)acetamido]cephalosporanic acid-(A) To a stirred solution of 5.35 g.(0.02 mole) of D'-()tx-(phydroxyphenyl) a (t butoxycarbonylamino)aceticacid, 2.02 g. (0.02 mole) of 2,6-lutidine and 50 ml. of tetrahydrofuranat l0 C. was added all at once 2.16 g. (0.02 mole) of ethylchloroformate. After 20 minutes an ice cold solution of 5.44 g. (0.02mole) of 7-aminocephalosporanic acid and 5 g. of sodium bicarbonate in50 ml. of water was added, all at once with vigorous stirring. Thetemperature was kept at or below 0 C. for 10 minutes and between 0 C.and +10 C. for minutes. Next, ml. of water was added and thetetrahydrofuran removed in vacuo at 20 C. One 200 ml. ether extract wastaken and discarded. The aqueous phase was layered with 200 ml. ofmethyl isobutyl ketone and cooled and stirred while being acidified topH 2. The methyl isobutyl ketone layer was washed with two 100 ml.portions of water, dried briefly over sodium sulfate, filtered andtreated with 7 ml. (0.2 mole) of 50% NaEI-l (sodium 2-ethylhexanoate inn-butanol). An oily precipate separated and slowly crystallized. Afterone hour, the. crystals were collected, washed with methyl isobutylketone and air dried. After further drying over phosphorus pentoxide(vacuum) there was obtained 5.24 g. dec. slowly above 100 C. Theinfrared and NMR spectra were consistent with the structure of sodium7-[D-u-(t-butoxy carbonylamino) a (p hydroxyphenyl) acetamido]cephalosporanate.

The free acid 7 D a (t butoxycarbonylamino)- 0c p hydroxyphenyl)acetamido]cephalosporanic acid was obtained as an amorphous gum byextracting an acidic aqueous solution with ethyl acetate andconcentrating under reduced pressure.

(b) Alternate procedure).-To a stirred solution of 5.35 g. (0.02 mole)of D a (p hydroxyphenyl)- a-(t-butoxycarbonylamino)acetic acid, 100 ml.of tetrahydrofuran, and 2.8 ml. (0.02 mole) of triethylamine at 40 C.was added dropwise 3.64 g. (0.02 mole) of trichloroacetic acid in 25 ml.of tetrahydrofuran over a 20 minute period. Next, after an additional 15minutes a solution of 5.44 g. (0.02 mole) of 7-aminocephalosporanic acidand 5.6 ml. (0.04 mole) of triethylamine in 300 ml. of methylenechloride precooled to 40 C. was added all at once and the temperaturemaintained at 40 C. to 30 C. for 45 minutes. The mixture was thenconcentrated under reduced pressure at 20 C. to an oil.

This was taken up in 200 ml. of 2% aqueous sodium bicarbonate and 200ml. of ether. The ether layer was discarded and the aqueous phaselayered with 200 ml. of ethyl acetate and, with cooling and stirring,the mixture acidified to pH 3. The ethyl acetate layer was thenseparated and washed twice with water, dried briefly over sodiumsulfate, filtered and evaporated to an oil under reduced pressure at 20C. Five hundred ml. of ether was then added and a small amount ofinsoluble material filtered 01f. The ether solution was thenconcentrated to about 200 ml. and then 200 ml. of Skellysolve B(petroleum ether) was added. The precipitate which formed was separatedby filtration and consisted of the desired product, 7 [D oz (tbutoxycarbonylamino) a (phydroxyphenyl)acetamido]cephalosporanic acid.Yield: 6 g.

7-[D-ot-amino-a- (p hydroxyphenyl) acetamido]cephalosporanic acid.7 [Du-(t-butoxycarbonylamino)-a- (p-hydroxyphenyl) acetamido]cephalosporanic acid (6 g.) was dissolved in 100 ml. of 50% aqueousformic acid and stirred at 40 C. for 3 hours. The solution was thentreated with 1 g. of decolorizing carbon and filtered. The filtrate wasconcentrated to a viscous oil at 20 C. under reduced pressure. The lasttraces of formic acid were removed by adding 300 ml. of toluene andremoving same under reduced pressure at 20 C. The resulting glass wastriturated with 400 ml. of ethyl acetate to which 5 ml. of water hadbeen added. A semi-crystalline solid formed which was filtered off andvacuum dried over phosphorus pentoxide. The product,7-[D-u-amino-a-(p-hydroxyphenyl)-acetamido]cephalosporanic acid, weighed1.8 g. and had a decomposition point of 260 C. with darkening above 150C. Infrared and NMR spectra were consistent with the structure.

Analysis.-Ca1cd for C18H19N3O7 (percent): C, 51.07; N, 4.55. Found(percent): C, 51.58; H, 5.12.

D()-a-benzyloxycarbonylamino a. (4 benzyloxycarbonylamino a (4benzyloxycarbonyloxyphenyl)- acetic acid.To a stirred suspension of 5.01g. (0.03 mole) of D-(-)-2-(p-hydroxyphenyl) glycine in 100 ml. of waterat 22 C. (room temperature) was added 1.2 g. (0.03 mole) of sodiumhydroxide pellets. A clear solution resulted. The stirred solution wascooled to C. and 2.4 g. (0.06 mole) of NaOH pellets were added. Whenthey had dissolved 13.6 g. (0.08 mole) of carbobenzoxy chloride wasadded all at once with vigorous stirring. After 30 minutes at 0 C. to 5C. the pH was 7 and a few drops of 50% NaAH-H O was added to keep the pHat 8-9 during another 30 minutes. Three hundred ml. of H 0 was thenadded and the resulting slurry was transferred to a separatory funneland 500 ml. of ether added. After shaking, the ether layer was discardedand the aqueous layer and solids combined with 300 ml. of ethyl acetateand the mixture acidified with shaking to pH 2 with 6 N HCl. The ethylacetate phase was combined with two more ethyl acetate extracts andwashed with two 100 ml. portions of water, two 300 ml. portions ofsaturated Na SO solution and filtered. Upon concentrating under reducedpressure to an oil the product crystallized. The material wasrecrystallized from benzene- Skellysolve B (pet. ether) to give 8.9 g.of D-( )-a-benzyloxycarbonylamino-a (4benzyloxycarbonyloxyphenyl)-acetic acid with a melting point of 101 102C.

Analysis.Calcd for C H NO (percent): C, 66.21; H, 4.88; N, 3.22. Found(percent): C, 67.93; H, 5.24; N, 3.07.

7- [D-a-amino-a- (p-hydroxyphenyl) acetamido]cephalosporanic acid.-D-a-benzyloxycarbonylamino-a- (4-benzyloxycarbonyloxyphenyl)acetic acid(0.344 mole) is dissolved in 100 mls. of dimethylformamide. There isthen added 2.6litidine (3.7 gms.; 0.044 mole) and the solution is cooledto 5 C. in an ice bath. Ethyl chloroformate (3.72 gms.; 0.0344 mole) isadded to the cool solution over a period of five minutes. The mixture isstirred for minutes and a solution of 7-aminocephalosporanic acid (0.395mole) in 70 ml. of water and 20 ml. of 2,6-lutidine is added. Thesolution is stirred in the ice bath for 15 minutes, diluted with 500 ml.of water and extracted twice with ether. The ether extract is discarded.The pH of the solution is lowered to 2 by the addition of dilute H andthe product is extracted into ether. The ether extract is washed withwater and the product is extracted into dilute Na CO This extract has apH of 7.5 and a volume of about 300 mls. It is then shaken with 7 gms.of 30% palladium on Celite for 20 minutes under an atmosphere ofhydrogen at a pressure of 50 p.s.i. The volume of the solution isdoubled by the addition of water and the pH is lowered to 2 by theaddition of dilute H 80 The catalyst is then removed by filtration andthe filtrate is extracted with a mixture of 150 mls. of methyl isobutylketone and 8 grns. of Aerosol O.T. The extract is dried over anhydrousNa SO and neutralized to pH 4.5 by the addition of triethylamine and anamorphous solid is collected by filtration and slurried with 20 mls. ofwater. A crystalline solid is formed which is collected and dried invacuo over P 0 This product, 7[D-a-amino-a-(p hydroxyphenyl)acetamido]cephalosporanic acid, is found to contain the fl-lactamstructure as shown by infrared analysis.

Celite is diatomaceous earth. Aerosol OT is dioctyl sodiumsulfosuccinate.

D-a-t-butoxycarboxamido 4 acetamidophenylacetic acid.A mixture ofD-aamino-4-acetamidophenylacetic acid (0.0205 mole), 3.2 g. (0.022 mole)of t-butoxycarbonyl azide, 1.65 g. (0.041 mole) of magnesium oxide andml. of 50% aqueous dioxane was stirred for 20 hours under a nitrogenatmosphere. The reaction mixture was poured into 400 ml. of ice waterplus 300 ml. of ethyl acetate. The ethyl acetate phase was twiceextracted with dilute aqueous sodium bicarbonate solution, the extractsbeing combined with the aqueous phase. The aqueous phase was acidifiedto pH 4 with 42% phosphoric acid and extracted with five 100-ml.portions of ethyl acetate. The combined and dried (sodium sulfate) ethylacetate extract was stripped of solvent at reduced pressure. A solutionof the residue in a minimum amount of chloroform was added to a largevolume of Skellysolve B petroleum solvent B.P. 60-68 C., essentiallyn-hexane) to yield 5.6 g. of. D-or-t-butoxycarboxamido-4-acetamidophenylacetic acid as an amorphous solid. The infrared and nuclearmagnetic resonance (NMR) spectra were consistent with the desiredproduct.

7 (D-u-amino 4 acetamidophenylacetamido)-cephalosporanic acid.A solutionof 5.6 g. (0.0182 mole) of D-a-t-butoxycarboxamido 4acetamidophenylacetic acid and 2.58 ml. (0.0182 mole) of triethylaminein 50 ml. of tetrahydrofuran was cooled to 45 C. (crystallization) and2.04 ml. (0.0182 mole) of trichloroacetyl chloride was added dropwiseduring 5 minutes at 45 C. After stirring for 10 minutes a cold (50)filtered solution of 4.96 g. (0.0182 mole) of 7-aminocephalosporanicacid and 5.1 ml. (0.0364 mole) of triethylamine in 250 ml. of methylenechloride was added in one portion. The reaction mixture was stirred at45 C. for one-half hour, then the cooling bath was removed and thetemperature allowed to rise to 0. The solvent was removed at reducedpressure. Water and ether were added to the residue. The ether phase wasextracted once with aqueous sodium bicarbonate solution and the extractcombined with the aqueous phase. The aqueous solution was acidified with42% phosphoric acid and extracted twice with ethyl acetate. The combinedethyl acetate extracts were twice washed with water, dried overanhydrous sodium sulfate and the solvent removed at reduced pressure.Trituration of the residue gave a solid which was dissolved inchloroform. Crystalline 7(D-wt-butoxycarboxamido-4-acetamidophenylacetamido)cephalosporanic acidseparated; weight 2.6 g.

7-(D-a-t-butoxycarboxamido 4 acetamidophenylacetamido)-cephalosporanicacid (2.45 g.) was added to 74 ml. of 45% aqueous formic acid and thesolution was stirred at 40 for 3 hours. The water and formic acid weredistilled off at reduced pressure, toluene finally being added anddistilled off to remove any remaining water and formic acid. The residuewas triturated with wet ethyl acetate, concentrated somewhat to removewater, finally more ethyl acetate was added giving a filterable solid.The solid was triturated with 95% ethanol yielding, after drying invacuo over phosphorus pentoxide, 1.4 g. of 7-(D-a-amino-4-acetamidophenylacetamido)-cephalosporanic acid. The infrared andnuclear magnetic resonance spectra were consistent with the desiredproduct.

D-a-t-butoxycarboxamido 3 acetamidophenylaceticacid.D-a-amino-3-acetamidophenylacetic acid (6.9 g., 0.0331 mole), 2.67g. (0.0662 mole) of magnesium oxide and 5.23 g. (0.0365 mole) oft-butoxycarbonyl azide were combined in 84 ml. of 50% aqueous dioxane.After 15 minutes of stirring, an additional 40ml. of 50% aqueous dioxanewas added. The mixture was stirred at 45 to 50 for 24 hours. Thereaction mixture was poured into 400 ml. of cold water, plus 300 m1. ofethyl acetate; the whole was then filtered to remove a small amount ofinsoluble material. The ethyl acetate phase was once extracted withdilute aqueous sodium bicarbonate and this combined with the aqueousphase. The cold aqueous solution was adjusted to pH 4 with 42%phosphoric acid and extracted with ethyl acetate. The ethyl acetateextract was washed three times with Water, dried with anhydrous sodiumsulfate and stripped of solvent at reduced pressure. The residue wasdissolved in anhydrous ether and diluted with Skellysolve B giving 5.9g. of D-ot-t-butoxycarboxamido-3- acetamidophenylacetic acid as anamorphous solid. The infrared and nuclear magnetic resonance spectrawere consistent with the desired product.

7-(Dm-t-butoxycarboxamido 3 acetamidophenylacetamido)-cephalosporanicacid.A cold to 5 C.) solution of 4.86 g. (0.0178 mole) of7-aminocephalosporanic acid and 2.5 ml. (0.0178 mole) of triethylaminein 25 ml. of water plus 25 ml. of tetrahydrofuran was added in oneportion to the vigorously stirred mixed anhydride prepared from 5.5 g.(0.0178 mole) of D-a-t-but0Xycarboxamido- 3-acetamidophenylacetic acid,2.5 ml. (0.0178 mole) of triethylamine and 1.7 ml. (0.018 mole) of ethylchloroformate in 75 ml. of tetrahydrofuran at 8 to 10 C. The mixture wasstirred at about 8" C. for 50 minutes then the cooling bath was removedand the mixture was stirred for 10 minutes. An additional 30 ml. ofwater was added. Most of the tetrahydrofuran was removed at reducedpressure. The aqueous concentrate was extracted three times with ethylacetate, ethyl acetate extracts being discarded. The aqueous phase wasacidified with 42% phosphoric acid and extracted three times with ethylacetate. A small amount of insoluble material was removed by filtrationduring the first extraction. The combined ethyl acetate extracts weredried with anhydrous sodium sulfate, the solvent removed at reducedpressure and the residue triturated with anhydrous ether giving 6.2 g.of 7 (D-a-t-butoxycarboxamido 3acetamidophenylacetamido)-cephalosporanic acid as an amorphous solid.The infrared and nuclear magnetic resonance spectra were consistent withthe desired product.

7 (D a amino 3 acetamidophenylacetamido)-cephalosporanic acid.Six g. of7 (D-u-t-blllOXYCEIIbOX- amido 3acetamidophenylacetamido)-cephalosporanic acid was added to 180ml. of44% aqueous formic acid. At first a solution was obtained and then acrystalline solid separated accompanied by gas evolution. The reactionmixture was stirred at 39 to 40 for 3 hours. The crystalline solid wasfiltered from the cooled reaction mixture and idtntified as 7-(D-a-t-butoxymrboxamido 3 acetamidophenylacetamido)-cephalosporanic acid,weight 2.1 g. having the following analysis (values corrected for 4.59%water): C, 53.6; H, 5.17; N, 10.08. Calcd. for C25H30N4O'9SI C, H, N,9.96.

The solvent was evaporated from the filtrate at reduced pressure.Toluene was added to the residue and this evaporated at reduced pressureto completely remove water and formic acid. This was repeated threetimes. The residue was triturated with wet ethyl acetate and then withanhydrous ether giving crude 7(D-a-arnino-S-acetamidophenylaeetamido)-cephalosporanic acid as afilterable solid; weight 3.8 g. This crude produce (3.5 g.) was combinedwith a two-phase system of water and ethyl acetate, a small amount ofdark colored impurity being removed by filtration. The filtrate wasconcentrated to a small volume at reduced pressure. The aqueousconcentrate deposited a small amount of crystalline impurity which wasremoved by filtration. The aqueous filtrate was concentrated to dryness,the residhe tritdrated with anhydrous ether and collected by filtrationgiving 1.6 g. of 7-(D-0L- amino 3acetamidophenylacetamido)-cephalosporanic acid. The infrared and nuclearmagnetic resonance spectra were consistent with the desired product.

D 0c t butoxycarboxamido-m-hydroxyphenylacetic acid.D aamino-m-hydroxyphenylacetic acid hydrobromide monohydrate (2.9 g.) in 25ml. of water was adjusted to pH 4.5 with aqueous sodium hydroxide.Dioxane (25 -ml.), 0.97 g. of magnesium oxide, and 3.43 g. oft-butoxycarbonyl azide were added and the mixture stirred overnight at30-40 C. under a blanket of nitrogen. The reaction mixture was pouredinto about 1 liter of ice water and 200 ml. of ethyl acetate. A smallamount of solid was removed by filtration. The filtrate was separatedinto its components and the ethyl acetate phase was extracted with 50ml. portions of 3% sodium bicarbonate solution. These extracts werecombined with the aqueous phase and the solution cooled and acidified topH 4.5 with 42% phosphoric acid. The product was extracted with five ml.portions of ethyl acetate and the combined extracts washed with waterand dried with anhydrous sodium sulfate.

The solvent was evaporated at reduced pressure giving 1.7 g. ofD-e-t-butoxycarboxamido-m-hydroxyphenylacetic acid as an oil.

An equivalent amount of either D-a-amino-m-hydroxy phenylacetic acid orthe hydrochloride monohydrate may be substituted for the amino acidhydrobromide monohydrate.

7 (D a t butoxycarboxamide-m-hydroxyphenylacetamido)-cephalosporanicacid.Ethyl chloroformate (0.57 ml., 0.006 mole) was added to a solutionprepared from 1.7 g. (0.006 mole) ofD-o-t-butoxycarboxamido-mhydroxyphenylacetic acid, 0.84 ml. (0.006 mole)of triethylamine and 50 ml. of tetrahydrofuran at 10 C. The mixture wasstirred at 10 C. for about 15 minutes to complete the formation of themixed anhydride. A cold (0) solution of 1.63 (0.006 mole) of7-aminocephalosporanic acid, 0.84 ml. (0.006 mole) of triethylamine, 25ml. of water, and about 25 ml. of tetrahydro furan was added to themixed anhydride and the reaction mixture stirred at 04 C. for one andone-half hours. The tetrahydrofuran was evaporated at reduced pressure.The aqueous residue was extracted once with ethyl acetate (discarded),then layered with ethyl acetate and acidified with 42% phosphoric acid.The aqueous phase was extraced with two additional portions of ethylacetate. The combined ethyl acetate extracts were washed with coldwater, dried with anhydrous sodium sulfate, and the solvent evaporatedat reduced pressure. The residue was triturated with Skellysolve B,benzene, and again with Skellysolve B. There was obtained, after drying,1.8 g. of 7 (Dtx-t-butoxycarboxamido-m-hydroxyphenylacetamido)cephalosporanic acid.The infrared spectrum was consistent with the desired product.

7 (D a-amino-m-hydroxyphenylacetamido)cephalosporanic acid.A solution of1.8 g. of 7-(D-a-t-butoxycarboxamido mhydroxyphenylacetamido)sephalosporanic acid in 53 ml. of 45% aqueousformic acid was stirred at 40 C. for three hours. The volatile materialswere evaporated at reduced pressure. The last traces of water and formicacid were removed by azeotropic distillation with toluene under reducedpressure. The oily residue was triturated with wet ethyl acetate givinga filterable solid. The solid was collected by filtration, washed withethyl acetate and dried in vacuo giving 890 mg. of7-(D-ocamino-m-hydroxyphenylacetamido)cephalosporanic acid. The infraredand nuclear magnetic resonance spectra were consistant with the desiredproduct.

Analysis.-Calcd. for C H O N S (percent): C, 51.30; H, 4.54; N, 9.97.Found (percent): C, 48.59; H, 5.02; N, 9.00; H O, 5.2. Found valuescorrected for 5.2% H C, 51.3; H, 4.4; N, 9.5.

D() a carbobenzoxyaminothienyl-3-acetic acid.- 5.0 g. (.0318 mole) ofD()-3-thienylglycine was dissolved in a solution of 2.5 g. (.0625 mole)of sodium hydroxide in 35 ml. of water. The resulting solution wascooled to 0 C. and 5.91 g. (.0348 mole) of carbobenoxy chloride wasadded dropwise with stirring. After the addition was completed, thereaction mixture was stirred for 2 hr. at 0-10 C. The solution wasextracted with 35 ml. of ether. The aqueous phase was acidified withconcentrated hydrochloric acid and a solid was removed by filtration.The filtrate was extracted with 2x50 ml. portions of ethyl acetate. Thecombined extracts were dried over anhydrous magnesium sulfate andevaporated to dryness to yield 7.36 g. (80%) ofD()-u-carbobenzoxyaminothienyl-3-acetic acid.

7 (D a. carbobenzoxyaminothienyl-3'-acetamido)- cephalosporanic acid.-Toa stirred suspensions of 7.25 g. (.0254 mole) ofD()-a-carbobenzoxyaminothienyl- 3-acetice acid in 120 ml. of drytetrahydrofuran containing 3.55 ml. (.0254 mole) of triethylamine, wasadded at 0-5" C. 3.0 g. (.0254 mole) of trimethylacetyl chloride. Afterbeing stirred for 8 minutes at 0 C. the mixture was treated with asolution containing 6.94 g. .0254 mole) of 7-aminocephalosporanic acid,7.1 ml. (0.508 mole) of triethylamine, 60 ml. of tetrahydrofuran and 60ml. of water. The reaction mixture Was then stirred for 40 minutes at 0C. and for 1 hr. at 0-8 C. and was then diluted with 200 ml. of water.The tetrahydrofuran was removed under reduced pressure, and the residuewas layered with ethyl acetate and acidified to pH 2.5 with 42%phosphoric acid. An emulsion formed, and this was filtered throughSuper-eel and the layers were separated. The aqueous phase was separatedand extracted with fresh ethyl acetate. The combined extracts werewashed with 50 ml. of saturated salt solution and were dried overanhydrous magnesium sulfate. Evaporation of the solvent afforded a solidwhich was triturated with dry ether. The product was collected byfiltration and dried to give 3.95 g. (28.4%) of 7 (Du-carbobenzoxyaminothienyl-3'- acetamido)cephalosporanic acid. It wascharacterized by its infrared spectrum (cm. (KBr disc): amide NH, 3300;carboxyl OH and adsorbed H O, 2400-3600; 6- lactam carbonyl, 1775;acetate, carboxyl, carbamate and amide carbonyls 1660-1725.

Sodium 7 (D-a-aminothienyl-3'-acetamido) cephalosporanate.A solutioncontaining 3.95 g. (.00725 mole) of 7 (D-a-carbobenzoxyaminothienyl 3'acetamidocephalosporanic acid, 40 ml. of 1,4-dioxane, 200 ml. of waterand 13 ml. of saturated sodium bicarbonate solution was shaken in anatmosphere of hydrogen at an initial pressure of 50 p.s.i. in thepresence of 3.98 g. of 30% palladium-on-diatomaceous earth catalyst formin. The reaction mixture was acidified to pH 2 with 6 N hydrochloricacid and was immediately filtered through Supercel to remove thecatalyst. To the filtrate was added saturated sodium bicarbonatesolution to bring the pH to 3.8. 250 ml. of n-butanol was also added andthe mixture was evaporated to dryness under reduced pressure. Theresidue was triturated with ether and the solid was collected and driedin vacuo. It was suspended in 5 ml. of methylene chloride containing0.28 ml. of triethylamine. The slurry was stirred for 15 min. and thenfiltered. To the filtrate was added 0.332 g. of sodium 2-ethylhexanoate(50% solution in ether) followed by 25 ml. of dry ether. The sodium saltwhich formed was collected by filtration, washed with dry ether anddried in vacuo over phosphorus pentoxide to afford a yield of 0.34 g. ofsodium 7-(D-aaminothienyl-3'-acetamido) cephalosporanate. The infraredspectrum (KBr disc) showed the following absorptions (emf): NH and NH,3200-3600; fi-lactam, and acetate carbonyls, 1760; amide carbonyl 1670;carboxylate, 1600.

Analysis.-Calcd. for C H N O S Na (percent): C, 44.34; H, 3.72. Found(percent): C, 44.21; H, 5.34.

Sodium D() a-[(1-carbomethoxypropene-Z-yl)amino]-thienyl-3-acetate.0.745g. (.0324 mole) of metallic sodium was dissolved in ml. of hot2-propanol. 5.10 g. .0324 mole) of methyl acetoacetate were added to thesolution, and the mixture was stirred and heated under reflux for 1 /2hr. The hot solution was filtered and the filtrate was allowed to coolto 5 C. The crystalline product which precipitated was collected byfiltration and dried in vacuo over phosphorus pentoxide to afford 7.0g., 78% of colorless needles of sodiumD()-a-[(1-carbomethoxypropene-Z-yl)aminoJthienyl-3-acetate, M.P. 209-212decomp. an analytical sample was prepared by recrystallization from hot2-propanol to give colorless rosettes of MJP. 215-217". Infraredspectrum (KBr disc. absorptions at 1645 CHI-1 for ester carbonyl and1575 cm." for carboxylate) and n.m.r. spectrum (solution in D 0) wereconsistent with the desired structure; the n.m.r. spectrum showed thesample contained 2-propanol.

Analysis.Calcd. for C H NO SNa-C H O (percent): C, 49.84; H, 5.98; N,4.15. Found (percent): C, 49.07, 48.85; H, 5.65, 6.04; N, 4.40.

7- [2',2'-dimethyl-5'-oxo-4- (3 "-thienyl)-1-imidazolidinyl]3acetoxymethyLM-cephem 4 carboxylic acid-A solution of 1.08 g. (0.1 mole)of ethyl chloroformate in 35 ml. of acetone containing 3 drops ofN,N-dimethyl benzylamine was cooled to 5 C. With stirring, 2.77 g. (.01mole) of powdered sodiumD()-a-[(1-carbomethoxypropene-2-yl)amino]thienyl-3-acetate was added,and the mixture was stirred for 20 min. at 5 to 0 C. A solution of 2.72g. .01 mole) of 7-aminocephalosporanic acid was prepared in 15 ml. ofwater by the dropwise addition of triethylamine to pH 8.0. 15 ml. ofacetone was added and the chilled solution was added with stirring tothe solution of the mixed anhydride previously described. After theaddition was completed, the mixture was stirred for 1 hr. at 0 C. It wasthen filtered and the acetone was removed by evaporation under reducedpressure. The residue was shaken briefly with 40 ml. of methyl isobutylketone and 5 ml. of 88% formic acid. The mixture was filtered throughSuper-cel and the Z-phase filtrate was stirred for 1 hr. at 0 C. andthen stored for 18 hr. at 0 C. An amorphous pale tan solid was collectedby filtration and dried in vacuo over phosphorus pentoxide. This processafforded 0.212 g., 5% of crude 7-[2,2-dimethyl- 5' oxo-4'(3"-thienyl)-1'-imidazolidinyl] 3 acetoxymethyl-A -cephem-4-carboxylicacid.

A suspension of 206 mg. (5 mole) of this material and 51 mg. (.5 mmole)of triethylamine in 5 ml. of dry acetone was stirred for 65 hr. at 23 C.The mixture was filtered and the filtrate was evaporated to dryness. Theresidue was suspended in water and the pH was adjusted to 3.5 with 20%phosphoric acid to give cream-colored, solid7-[2,2-dimethyl-5'-oxo-4'-(3"-thienyl)-1-imidazolidinyl]-3-acetoxymethyl-A-cephem 4 carboxylic acid, which was collected by filtration, washedwith cold water and dried in vacuo over phosphorus pentoxide. Theinfrared spectrum (KBr disc.) had absorption (emffor NH, 3350; fi-lactamcarbonyl, 1780; acetate, carboxyl and imidazolidinone carbonyls,1740-1700, acetate, 1230.

D -a-t-butoxycarboxamido-3-thienylacetic acid.An intimate mixture of15.72 g. (0.1 mole) of D()-3-thienylglycine and 8.06 g. (0.2 mole) ofmagnesium oxide powder was suspended in 250 ml. of 50% aqueous dioxane.

28.6 g. (0.2 mole) of t-butoxycarbonyl azide was added dropwise withvigorous stirring, and the mixture was stirred for 20 hr. at 45-50. Themixture was cooled, diluted with 1 l. of ice-water and stirred brieflywith 250 ml. of ethyl acetate. The layers were separated and the organicphase was extracted with 50 ml. of 3% sodium bicarbonate solution andthen with 50 ml. of water. All the aqueous fractions were then combinedand the pH was lowered to with 42% phosphoric acid. The product wasextracted into 3X 250 ml. portions of ethyl acetate. The combinedextracts were washed with water, dried over magnesium sulfate, andevaporated to dryness under reduced pressure. Trituration of the oilyresidue with ether and Skellysolve B gave 18.7 g. (72%) of crystallineD()-a-t-butoxycarboxamido-S-thienylacetic acid M.P. l02103. Ananalytical sample was recrystallized from ethyl acetate and SkellysolveB to give colorless needles, M.P. 104105, [051 -107 (C=1.0, CH OH).

Calcd. for C H NO S (percent): C, 51.36; H, 5.88. Found (percent): C,51.49; H, 5.56.

7-(D-a t butoxycarboxamido 3 thienylacetamido)- cephalosporanic acid.Asolution of 12.87 (.05 mole) ofD()-a-t-butoxycarboxamido-3-thienylacetic acid and and 5.06 g. (.05mole) of triethylamine in 200 ml. of dry tetrahydrofuran was cooled to-10. 5.43 g. (.05 mole) of ethyl chloroformate was added dropwise withstirring and the resulting mixed anhydride solution was stirred for 12min. at 10. A chilled (5) solution of 13.62 g. (.05 mole) of7-aminocephalosporanic acid and 5.06 g. (.05 mole) of triethylamine in200 ml. of 50% aqueous tetrahydrofuran was added in one portion. Thereaction mixture was stirred for 1% hr. at 0 and after filtration thetetrahydrofuran Was removed from the filtrate under reduced pressure.The residue was dissolved in a mixture of 300 ml. of Water and 150 ml.of ethyl acetate. After a brief shaking, the layers were separated andthe aqueous phase was cooled and acidified to pH 3 with 42% phosphoricacid. The product was extracted into 3 X 200 ml. portions of ethylacetate and the combined extracts were Washed with water and dried overmagnesium sulfate. Evaporation of the solvent left an oil which wassolidified by trituration with ether and Skellysolve B to give 21.7 g.(85%) of 7-(D-u-t-butoxycarboxamido-3-thienylacetamido)cephalosporanicacid. It was characterized by its infrared spectrum (cmf (KBr disc):amide NH, 3340, fi-lactam carbonyl, 1790 acetate, amide, carbamate andcarboxyl carbonyls, 17504680; amide deformation 1520.

7-(D a amino 3 thienylacetamido)cephalosporanic acid.A solution of 7.67g. (.015 mole) of 7-(D-a-tbutoxycarboxamido 3 thienylacetamido)cephalosporanic acid in 400 ml. of 45% aqueous formic acid Was stored at40 for 3 hr. The solution was evaporated to dryness under reducedpressure and the last traces of formic acid were removed by azeotropicdistillation with toluene under reduced pressure. The residue wastriturated with a mixture of 40 ml. of ethyl acetate and 2 ml. of water,and then with ether to give 5.3 g. (86%) of 7- (D-oc amino 3thienylacetamido)cephalosporanic acid. Infrared and n.m.r. spectra werefully consistent with the structure.

Calcd. for C H N O S- /2H O (percent) C, 49.66; H, 4.46. Found(percent): C, 44.54; H, 4.83.

7-[2,2-dimethyl-5-oxo-4-(3-thienyl)-1 imidazolidinyl] cephalosporanicacid-5.1 g. of 7-(D-0c-3IhiI10-3-fl1i6I1Ylacetamido)cephalosporanic acidwas slurried in a mixture of 170 ml. of acetone and 130ml. of methanol.1.23 g. (1 equivalent) of triethylamine was added with stirring and theresulting solution was stirred for 16 hr. at room temperature. A smallamount of insoluble material was removed by filtration and the filtratewas evaporated to dryness under reduced pressure. The residue wasdissolved in a mixture of 60 ml. of water and 40 ml. of ethyl acetate.This mixture was cooled and the pH of the aqueone phase was lowered to310 with 42% phosphoric acid with stirring. The layers were separatedand the aqueous phase was extracted with 2 x 60 ml. of fresh ethylacetate. The combined extracts were washed with water and evaporated todryness, and the solid residue was triturated with ether to give 2.1 g.(38%) of hydrated 7-[2,2-dimethyl-5-oxo-4-(3 thienyl) 1 imidazolidinyl]cephalosporanic acid. Infrared and n.m.r. spectra Were fully consistentwith the proposed structure.

Calcd. for C H N O S- A2H O (percent: C, 49.66; H, 4.61. Found(percent): C, 49.48, H, 4.83.

The synthesis of desacetoxycephalosporins, including cephalexin and someanalogs, has also been described by C. W. Ryan et al., J. Med. Chem. 12,310313 (March 1969) and in the references given in the footnotestherein.

I claim:

1. A compound of the formula wherein Ar is 2-thienyl, 3-thienyl or inwhich each of R and R is hydrogen, nitro, di(lower)- alkylamino, (lower)alkanoylamino, amino, hydroxy, (lower)alkanoyloxy, (lower)alkyl,(lower)alkoxy, sulfamyl, chloro, iodo, bromo, fiuoro or trifluoromethylor a nontoxic, pharmaceutically acceptable salt thereof.

2. A compound as claimed in claim 1 wherein Ar represents phenyl.

3. A compound as claimed in claim 1 wherein Ar represents 2-thienyl.

4. A compound as claimed in claim 1 wherein Ar represents 3-thienyl.

5. A compound as claimed in claim 1 wherein Ar representsp-hydroxyphenyl.

6. 3-azidomethyl 7 (D 0c aminophenylacetamido) ceph-3-em-4-oic acid.

7. The sodium salt of the compound of claim 6.

8. The potassium salt of the compound of claim 6.

9. An inorganic acid addition salt of the compound of claim 6.

10. 3-azidomethyl-7-(D-a amino 2 thienylacetamido)-cep-3-em-4-oic acid.

11. The sodium salt of the compound of claim 10.

12. The potassium salt of the compound of claim 10.

13. 3-azidomethyl-7 (D 0c amino 3 thienylacetamido)-ceph-3-em-4-oicacid.

14. The sodium salt of the compound of claim 13.

15. The potassium salt of the compound of claim 13.

16. A compound as claimed in claim 1 wherein Ar representsacetamidophenyl.

17. A compound as claimed in claim 1 wherein Ar representsm-acetamidophenyl.

18. 3-azidomethyl-7 (D 0c amino macetamidophenylacetamido)ceph-3-em-4-oic acid.

19. The sodium salt of the compound of claim 18.

20. The potassium salt of the compound of claim 18.

References Cited UNITED STATES PATENTS 3,497,505 2/ 1970 Pfeiffer et al260243 C NICHOLAS S. RIZZO, Primary Examiner US. Cl. X.R.

